Automated system for sample preparation and analysis

ABSTRACT

A sample preparation and analysis system. The system includes a sample preparation system and a sample analysis system. The sample preparation system prepares samples in accordance with an assay that is selected from a database containing a plurality of unique assays. The sample analysis system includes an analyzer that is dynamically reconfigurable based on the selected assay so as to analyze the prepared sample in accordance with that selected assay. A data communication link communicates data from the sample preparation system to the sample analysis system to reconfigure the analyzer in accordance with the selected assay.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.13/882,393, filed Jul. 17, 2013, which is a submission under 35 U.S.C.§371 of International Application No. PCT/US2011/058452, filed Oct. 28,2011, which claims the benefit of U.S. Provisional Application Ser. No.61/408,180, filed Oct. 29, 2010, the disclosures of which are herebyexpressly incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to the field of samplepreparation and analysis and, more particularly, to sample preparationsystems and sample analysis systems for preparing and analyzing samplesaccording to a variety of different analyte assays.

BACKGROUND OF THE INVENTION

Liquid chromatography mass spectrometry (“LCMS”) is a powerful analytedetection and measurement technique that has become the preferred methodof detecting small molecule, amino acid, protein, peptide, nucleic acid,lipid, and carbohydrate analytes to a high accuracy for diagnosticpurposes. However, the instrumentation required for LCMS is technicallycomplex and not well suited to the typical hospital clinical lab ormedical lab technician. These clinical labs have not adopted LCMSdiagnostics and, instead, generally use alternative diagnostictechniques, including automated immunoassay. Alternatively, the clinicallabs may send the samples out to a central reference laboratory foranalysis.

Current LCMS methods require careful selection of the appropriate liquidchromatography column and mobile phases for each analyte assay, as wellas complex calibration of the mass spectrometer to isolate and identifythe analyte of interest. Moreover, in order to analyze a differentanalyte or different class of analyte on the same instrument, one ormore of the column, the mobile phases, the liquid chromatographysettings, and/or the mass spectrometer settings must be changed andoptimized by the LCMS technologist. Often, individual hardwarecomponents, such as the ion source of the mass spectrometer, must bemanually re-configured in order to accommodate a different mode ofanalysis for a particular analyte. Such complicated equipment andsophisticated scientific techniques require very sophisticated LCMSspecialist technologists, and heretofore only the large centrallylocated reference laboratories have been able to use such LCMS equipmentfor clinical diagnostics.

At each such reference laboratory, because of the time and technicalcomplexity of such equipment adjustments, patient specimens that utilizethe same type of assay are generally grouped into large batches andprocessed serially, in order to avoid the necessity of making manualadjustments that are time consuming and may be prone to produce errors.While this batch mode automation approach may reduce the amount of LCMStechnologist intervention, it significantly increases the “time toresult” for each specimen. Thus, non-urgent specimens may not beprocessed for several hours or even days prior to analysis. For timesensitive specimens, for example, for emergency department patients ortransplant patients requiring short turnaround time results forimmediate treatment decisions, such delays are unacceptable.

Still further, some specimens have a limited shelf-life due todeterioration of one or more analytes or evaporation which distorts theconcentration of the analyte. Therefore, there is a set of complexfactors that determine how long a specimen of a particular type may bedelayed behind other specimens of a higher priority.

For a typical hospital lab, an LCMS system is a very large capitalinvestment. As such, it is often impractical for a hospital lab topurchase multiple systems for different dedicated analyses. Thus, it isimpractical for a hospital, even a large one, to use batch modeautomation for clinical LCMS application, as it does not have the scaleof a central reference laboratory and may be forced to make frequentchanges to the LCMS hardware and complex setting, as it switches thetesting from one type or class of analytes to another. Faced with thelarge economic and technical challenges, such clinical labs have beenunable to reap the technical benefit of LCMS technologies for routinepatient specimens, but have been forced to send patient samples on tothe central reference labs.

Therefore, there is a need for sample preparation and sample analysissystems that are more flexible for handling different types of analyteassays. There is also a need for sample preparation and sample analysissystems that are less complex to configure and use for preparing samplesand conducting a variety of different analyte assays, without requiringthe expertise of LCMS technologists, or the massive scale of a referencelaboratory. There is yet also a need for a sample preparation and sampleanalysis systems that improve the efficiency of the time to result for avariety of different analyte assays.

SUMMARY OF THE INVENTION

The present invention overcomes the foregoing problems and othershortcomings, drawbacks, and challenges of conventional samplepreparation and sample analysis systems. While the invention will bedescribed in connection with certain embodiments, it will be understoodthat the invention is not limited to these embodiments. To the contrary,this invention includes all alternatives, modifications, and equivalentsas may be included within the spirit and scope of the present invention.

In accordance with one embodiment of the present invention, a samplepreparation and analysis system includes a sample preparation system anda sample analysis system. The sample preparation system prepares samplesin accordance with an assay that is selected from a database containinga plurality of unique assays. The sample analysis system includes ananalyzer that is dynamically reconfigurable based on the selected assayso as to analyze the prepared sample in accordance with that selectedassay. A data communication link communicates data from the samplepreparation system to the sample analysis system to reconfigure theanalyzer in accordance with the selected assay.

According to another embodiment of the present invention, a method ofpreparing and analyzing a sample includes preparing a sample with asample preparation system and analyzing the prepared sample with asample analysis system. The sample is taken from a specimen inaccordance with an assay that is selected from a database containing aplurality of unique assays. The sample analysis system includes ananalyzer, which is dynamically reconfigured in response to the selectedassay and analyzes the prepared sample in that regard. Data todynamically reconfigure the analyzer is communicated between the samplepreparation system and the sample analysis system over a datacommunication link.

Another embodiment of the present invention is directed to a samplepreparation and analysis system that includes a sample preparationstation and a sample analysis station. The sample preparation systemprepares samples in accordance with an assay that is selected from adatabase containing a plurality of unique assays. The sample analysissystem includes an analyzer that is dynamically reconfigurable based onthe selected assay so as to analyze the prepared sample in accordancewith that selected assay. A transport mechanism transports the preparedsample from the sample preparation system to the sample analysis system.

Still another embodiment of the present invention is directed to amethod to prepare and analyze a sample by a sample preparation systemand sample analysis system, respectively. The sample is taken from aspecimen in accordance with an assay that is selected from a databasecontaining a plurality of unique assays. The sample analysis systemincludes an analyzer, which is dynamically reconfigured in response tothe selected assay and analyzes the prepared sample in that regard. Atransport mechanism transports the prepared sample from the samplepreparation system to the sample analysis system.

In accordance with another embodiment of the present invention, anencapsulated sample preparation and analysis system having a samplepreparation system and a sample analysis system includes a controller.The controller controls the operation the sample preparation system aswell as the operation of a portion of the sample analysis system.Operation of the sample preparation system and the portion of the sampleanalysis system are in accordance with an assay, which is selected froma database containing a plurality of unique assays.

According to another embodiment of the present invention, anencapsulated sample preparation and analysis system includes acontroller. The controller is configured to dynamically andautomatically vary one or more parameters of a sample preparation systemin order to prepare samples in accordance with respective assays. Theassays are selected from a database containing a plurality of uniqueassays.

Yet another embodiment of the present invention is directed to anautomated sample preparation and analysis system that includes a samplepreparation system and a sample analysis system. The sample preparationstation prepares a sample, taken from a specimen, in accordance with anassay that is selected from a plurality of unique assays. The sampleanalysis system is configured to analyze the prepared sample accordingto the selected assay. Sequencing of the preparation of the samples inthe sample preparation system and analysis by the sample analysis systemis controlled by a controller.

Still another embodiment of the present invention includes a method ofpreparing and analyzing samples taken from specimens. The methodincludes sequencing the samples for preparation, which is in accordancewith respective assays selected from a database containing a pluralityof unique assays. The method further includes sequencing the analysis ofthe prepared samples, which is also in accordance with the respectiveassays.

In accordance with another embodiment of the present invention, anautomated sample preparation and analysis system includes a samplepreparation system and a sample analysis system. The sample preparationsystem prepares a sample taken from a specimen in accordance with anassay, which is selected from a database containing a plurality ofunique assays. The sample analysis system is configured to analyze theprepared sample in accordance with the selected assay. A controllerdynamically sequences the prepared sample for analysis by the sampleanalysis system.

Another embodiment of the present invention is directed to a method ofpreparing and analyzing samples taken from specimens. The methodincludes dynamically sequencing the analysis of the prepared samplesaccording to respective assays selected from a database containing aplurality of unique assays.

Another embodiment of the present invention is directed to a method ofpreparing and analyzing samples. The method includes querying a firstcontroller with a second controller to determine a plurality of uniqueassays that may be performed by the sample analysis station. A humanperceptible indication of the plurality of unique assays is provided.

Yet another embodiment of the present invention is directed to a methodof preparing a sample by receiving a specimen and automaticallydetermining a test to be performed on the sample. The test is inaccordance with an assay selected from a database containing a pluralityof unique assays. A plurality of preparation steps is determined inresponse to the selection of the assay. The preparation steps preparethe sample for analysis.

Still another embodiment of the present invention is directed to amethod of preparing a sample. The method includes receiving a specimenand then, automatically, determining a target time to prepare a samplefrom the specimen. The target time to prepare is indicative of a time atwhich preparation of the sample should be complete.

In accordance with another embodiment of the present invention, a methodof analyzing a sample includes receiving a specimen. The sample isprepared from the specimen and then, automatically, determining a targettime to result. The target time to result is indicative of a time atwhich the analysis of the prepared sample is returned.

In accordance with yet another embodiment of the present invention, amethod of multiplexing the operation of a sample preparation andanalysis system include preparing first and second prepared samples witha sample preparation station. The first prepared sample is prepared inaccordance with a first assay, and the second prepared sample isprepared in accordance with a second assay. The first and second assaysare selected from a database containing a plurality of unique assays.The first and second prepared samples are transported, separately, to asample analysis station having first and second separation channels andan analyzer. The first prepared sample is analyzed in accordance withthe first selected assay. While analyzing the first prepared sample, thesecond prepared sample is being separated with the second separationchannel and in accordance with the second selected assay.

According to still another embodiment of the present invention, a methodof multiplexing a sample preparation and analysis system having a samplepreparation system and a sample analysis system includes determining asample analysis system readiness. The sample analysis system isconfigured to analyze a plurality of prepared samples with a massspectrometer. The injection of the plurality of prepared samples intoone a plurality of injection ports is sequenced. Each of the pluralityof injection ports is coupled to a respective separation channel. Thesequencing is in accordance with information associated with therespective one of the plurality of prepared samples and the sampleanalysis system readiness.

According to another embodiment of the present invention, a method ofmultiplexing a sample preparation and analysis system having a samplepreparation system and a sample analysis system includes receiving aspecimen at the sample preparation system. An indication of an assayassociated with the specimen is automatically determined such that anassay corresponding to the indication of the assay is selected from adatabase containing a plurality of unique assays. A sample is taken fromthe specimen in accordance with the selected assay with the samplepreparation system. A mass spectrometer and/or a separation channel ofthe sample analysis system are dynamically reconfigured according to theselected assay. The reconfigured sample analysis system processes theprepared sample according to the selected assay.

One embodiment of the present invention is directed to a samplepreparation and analysis system that is configured to inhibitevaporation of volatile liquids used therein. The sample preparation andanalysis system includes a sample preparation system that is configuredto receive a plurality of openable sample vessels, wherein each of theopenable sample vessels is configured to be opened and to receive asample and/or a volatile liquid therein. The openable sample vessel isthen closed to inhibit evaporation of the volatile liquid.

Another embodiment of the present invention is directed to a samplepreparation and analysis system. That includes a sample preparationstation and a sample analysis station. The sample preparation stationprepares a sample taken from a specimen for analysis in accordance withan assay. The assay is selected from a database containing a pluralityof unique assays. The sample preparation system includes a firstcontroller to control at least a portion of the operation of the samplepreparation system. The sample analysis system analyzes the preparedsample using an analyzer configured in accordance with the selectedassay. The sample analysis system further includes a second controllerto control at least a portion of the operation of the sample analysissystem. A software data communication link and a hardware datacommunication link each communicate data between the first and secondcontrollers.

Still another embodiment of the present invention is directed to amethod of preparing and analyzing a sample. The method includespreparing a sample for analysis by mass spectrometry by a samplepreparation system. The sample is prepared in accordance with an assayselected from a database containing a plurality of unique assays. Thesample preparation system includes a first controller to control atleast a portion of the operation of the sample preparation system. Amass spectrometer analyzes the prepared sample in accordance with theselected assay and includes a second controller to control at least aportion of the operation of the mass spectrometer. Data associated withthe prepare sample is communicated between the first and secondcontrollers via at least a portion of a data communication link, whichincludes a software data link and a hardware data link.

Yet another embodiment of the present invention is directed to anautomated sample preparation and analysis system having a samplepreparation system and a sample analysis system. The sample preparationsystem prepares a sample for an assay, which is selected from a databasecomprising a plurality of unique assays. The sample analysis systemincludes a mass spectrometer for analyzing the prepared sample inaccordance with the selected assay. First and second controllers areconfigured to control at least a portion of the sample preparationsystem and the sample analysis system, respectively. The secondcontroller is further configured to send result data to the firstcontroller.

In accordance with another embodiment of the present invention, a methodto prepare and analyze a sample includes preparing a sample for an assaywith a sample preparation system. The assay is selected from a databasecontaining a plurality of unique assays. A sample analysis system, whichincludes a mass spectrometer, analyzes the prepared sample according tothe selected assay. Data with respect to at least one of a result of theanalysis and an identification of the prepared sample is communicatedfrom the sample analysis system to the sample preparation system.

According to another embodiment of the present invention, an automatedsample preparation and analysis system includes a sample analysis systemand a sample preparation system. The sample analysis system includes amass spectrometer and is configured to analyze a plurality of samplesaccording to respective assays selected from a database containing aplurality of unique assays. The sample preparation system includes acontroller for sequencing the samples for analysis by the sampleanalysis system. The sequence of the sample analysis is dependent on theorder of arrival to the automated sample preparation and analysissystem, the priority status of each sample, and at least one of a targettime to result for a sample, a target time to result for a selectedassay, a remaining target time to result for a selected assay, a numberof samples on-board, the number of samples awaiting analysis inaccordance with the same selected assay, or mass spectrometerreconfiguration necessary for selected assays that precede and follow aselected assay for a particular sample.

In accordance with another embodiment of the present invention, anautomated biological specimen preparation and mass spectrometry analysissystem for analyzing a plurality of biological specimens according to aselected assay from a database containing a plurality of unique assaysincludes a sample preparation system and a sample analysis system. Thesample preparation system prepares samples taken from at least one ofthe plurality of biological specimens. The sample analysis system, witha mass spectrometer, quantifies one or more analytes for one or moreprepared samples. A specimen dock receives a plurality of containers,each containing a respective biological specimen. A reagent stationreceives a plurality of containers containing a reagent liquid. A samplestation transfers a predetermined biological specimen and one or morepredetermined reagent liquids to a sample vessel. An analysis stagingstation stores one or more sample vessels, each is containing arespective prepared sample. A transport mechanism transfers the preparedsamples from one of the sample vessels to the sample analysis system.

Still another embodiment of the present invention is directed to amethod of entering an idle state for a sample preparation and analysissystem. The sample preparation and analysis system includes a samplepreparation system and a sample analysis system. To enter the idlestate, the sample preparation and analysis system receives an idle statecommand. A plurality of blank samples is prepared by the samplepreparation system in accordance with an assay that is selected from adatabase containing a plurality of unique assays. The blank samples donot include a sample of a specimen. First and second ones of theplurality of blank samples are analyzed with the sample analysis systemin accordance with the selected assay. Other ones of the plurality ofblank samples are analyzed with the sample analysis system in accordancewith the selected assay until a wake up command is received.

Another embodiment of the present invention is directed to a method ofentering a standby state for a sample preparation and analysis system.The sample preparation and analysis system includes a sample preparationsystem and a sample analysis system. To enter the standby state, thesample preparation and analysis system receives a standby state command.A standby sample is prepared by the sample preparation system inaccordance with an assay that is selected from a database containing aplurality of unique assays and does not include a sample of a specimen.The standby sample is analyzed with the sample analysis system inaccordance with the selected assay. At least one component of the samplepreparation and analysis system is then powered down. The at least onecomponent is selected from a group comprising a chromatography columnheater, a gas flow, a temperature of an ionization source of a massspectrometer, at least one vacuum pump, at least one fluid pump, arobotic device, a pipette assembly, a mixing station, an incubationstation, a matrix interference removal station, a cooling system, and aheating system.

The above and other objects and advantages of the present inventionshall be made apparent from the accompanying drawings and thedescriptions thereof.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with the general description given above and the detaileddescription of the embodiments given below, serve to explain theprinciples of the present invention. In the figures, corresponding orlike numbers or characters indicate corresponding or like structures.

FIG. 1A is a perspective view of an automated sample preparation andanalysis system in accordance with one embodiment of the presentinvention.

FIG. 1B is a perspective view of an automated sample preparation andanalysis system in accordance with another embodiment of the presentinvention.

FIG. 2 is a diagrammatic view of the automated sample preparation andanalysis system of FIG. 1A.

FIG. 3A is a top view of the automated sample preparation and analysissystem of FIG. 1A and according to one embodiment of the presentinvention.

FIG. 3B is a top view of the automated sample preparation and analysissystem of FIG. 1B and according to one embodiment of the presentinvention.

FIG. 3C is a side elevational view of the automated sample preparationand analysis system of FIG. 1A with the front cover removed.

FIG. 3D is a side elevational view of the automated sample preparationand analysis system of FIG. 1B with the front cover removed.

FIG. 4A is a schematic view of a sample preparation station and atransport assembly of the automated sample preparation and analysissystem of FIG. 1A in accordance with one embodiment of the presentinvention.

FIG. 4B is a schematic view of a sample preparation station and a sampleanalysis station of the automated sample preparation and analysis systemof FIG. 1A in accordance with one embodiment of the present invention.

FIGS. 5A and 5B are side elevational views of a reagent container inaccordance with one embodiment of the present invention, shown in closedand open states, respectively.

FIGS. 6A and 6B are side elevational view of a sample vessel inaccordance with one embodiment of the present invention, shown in closedand open states, respectively.

FIG. 7A is a side elevational view of a dual-column chromatographycartridge in accordance with one embodiment of the present invention.

FIG. 7B is a side elevational view of a dual-column chromatographycartridge in accordance with another embodiment of the presentinvention.

FIG. 7C is a perspective view of a top portion of a dual-columnchromatography cartridge in accordance with one embodiment of thepresent invention.

FIG. 7D is a perspective view of a bottom portion of a dual-columnchromatography column in accordance with another embodiment of thepresent invention.

FIG. 8 is a perspective view of a cradle configured to receive one ormore dual-column chromatography cartridges in accordance with oneembodiment of the present invention.

FIG. 9A is a perspective view of a pump for a dual-column liquidchromatography of an automated sample preparation and analysis system inaccordance with one embodiment of the present invention.

FIG. 9B is a cross-sectional view taken along line 9B-9B in FIG. 9A,with a piston of the pump retracted.

FIG. 9C is a cross-sectional view taken along line 9B-9B in FIG. 9A,with the piston of the pump extended.

FIG. 9D is a cross-sectional view taken along line 9D-9D in FIG. 9B.

FIG. 9E is a cross-sectional view taken along line 9E-9E in FIG. 9C.

FIG. 9F is a cross-sectional view of a portion of a pump in accordancewith another embodiment of the present invention.

FIG. 9G is a cross-sectional view taken along line 9G-9G in FIG. 9F.

FIG. 10A is a schematic illustration of a multi-port valve having afill-in loop, shown in an “in-line” position, and according to oneembodiment of the present invention.

FIG. 10B is a schematic illustration of the multi-port valve of FIG.10A, shown in a “fill in loop” position.

FIG. 11 is a diagrammatic view of a hardware and software environmentfor a sample preparation controller and in accordance with oneembodiment of the present invention.

FIG. 12 is a diagrammatic view of a plurality of modules for a samplepreparation controller and in accordance with one embodiment of thepresent invention.

FIG. 13 is a diagrammatic view of the data structure included in themass storage device of a sample preparation controller in accordancewith one embodiment of the present invention.

FIG. 14 is a diagrammatic view of a hardware and software environmentfor a sample analysis controller and in accordance with one embodimentof the present invention.

FIG. 15 a diagrammatic view of a plurality of modules for a sampleanalysis controller and in accordance with one embodiment of the presentinvention.

FIGS. 16A-16G are exemplary screenshots provided by a sample preparationcontroller in accordance with one embodiment of the present invention.

FIG. 17 is a flowchart illustrating a sequence of the operations forcollecting, preparing, and analyzing a prepare sample in accordance withone embodiment of the present invention.

FIG. 18 is a flowchart illustrating a sequence of operations forpreparing a sample in accordance with one embodiment of the presentinvention.

FIG. 19 is a flowchart illustrating a sequence of operations forpreparing a calibration and/or control standard in accordance with oneembodiment of the present invention.

FIG. 20 is a flowchart illustrating a sequence of operations forpreparing a specimen with an appropriate time to result in accordancewith one embodiment of the present invention.

FIG. 21 is a flowchart illustrating a sequence of operations for ascheduler to determine the steps of preparing a sample according to oneembodiment of the present invention.

FIG. 22 is a flowchart illustrating a sequence of operations fordetermining whether a particular assay may be performed and according toone embodiment of the present invention.

FIG. 23 is a flowchart illustrating a sequence of operations forperforming a calibration check in accordance with one embodiment of thepresent invention.

FIG. 24 is a flowchart illustrating a sequence of operations forperforming or overriding a particular test according to one embodimentof the present invention.

FIG. 25 is a flowchart illustrating a sequence of operations for acentrifuge and a mixer according to one embodiment of the presentinvention.

FIGS. 26A-26C are a flowchart illustrating a sequence of operations forinjecting a prepared sample into an analysis station and in accordancewith one embodiment of the present invention.

FIG. 27A-27B are a flowchart illustrating a sequence of operations fordetermining and performing an assay type according to one embodiment ofthe present invention.

FIG. 28 is a flowchart illustrating a sequence of operations forbuilding a methodology for performing a sample analysis according to oneembodiment of the present invention.

FIG. 29 is a flowchart illustrating a sequence of operations to collectdata for an appropriate analyte according to one embodiment of thepresent invention.

FIG. 30 is a flowchart illustrating a sequence of operations to monitora status of the sample analysis station according to one embodiment ofthe present invention.

FIG. 31 is a flowchart illustrating a sequence of operations forprocessing and reporting various sample types according to oneembodiment of the present invention.

FIG. 32 is a flowchart illustrating a sequence of operations toconsolidate vessels in vessels racks according to one embodiment of thepresent invention.

FIG. 33 is a flowchart illustrating a sequence of operations todetermine whether to discard specimens according to one embodiment ofthe present invention.

FIGS. 34A-34B are a flowchart illustrating a sequence of operations tobooting an automated sample preparation and analysis system inaccordance with one embodiment of the present invention.

FIGS. 35A-35B are a flowchart illustrating a sequence of operations forstarting and entering an idle state of an automated sample preparationand analysis system in accordance with one embodiment of the presentinvention.

FIG. 36 is a flowchart illustrating a sequence of initiating a standbystate for an automated sample preparation and analysis system inaccordance with one embodiment of the present invention.

FIG. 37 is a flowchart illustrating a sequence of shutting down anautomated sample preparation and analysis system in accordance with oneembodiment of the present invention.

FIG. 38 is a fluid system for managing one or more fluid levels withinan automated sample preparation and analysis system in accordance withone embodiment of the present invention.

FIG. 39A is an exemplary chromatograph for a second sample prepared andanalyzed with an automated sample preparation and analysis system and inaccordance with an embodiment of the present invention.

FIG. 39B is exemplary raw data acquired from an automated samplepreparation and analysis system, shown in a tabular format.

FIG. 39C is an exemplary linear response of an ion detector for a firstanalyte analyzed by an automated sample preparation and analysis systemin accordance with one embodiment of the present invention.

FIG. 39D is an exemplary negative exponential response of an iondetector fit to the response of an ion detector for a second analyteanalyzed by an automated sample preparation and analysis system inaccordance with one embodiment of the present invention.

FIG. 39E is an exemplary positive exponential response of an iondetector fit to the response of an ion detector for a third analyteanalyzed by an automated sample preparation and analysis system inaccordance with one embodiment of the present invention.

FIG. 40 is an exemplary graphical view of a total ion current forvarious m/z values measured at an ion detector of an automated samplepreparation and analysis system in accordance with one embodiment of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1A is a perspective illustration of an automated sample preparationand analysis system 10 according to one exemplary embodiment of thepresent invention (referred to hereinafter as “system” 10). The system10 is designed to automatically prepare a sample from a specimen foranalysis and to analyze the prepared sample according to a predeterminedanalyte assay selected from a variety of different or unique analyteassays. As will be described in greater detail below, the exemplarysystem 10 is particularly designed to perform two distinct laboratoryfunctions, i.e., sample preparation and sample analysis, in combinationin an automated system.

FIG. 1B, like FIG. 1A, is a perspective illustration of an automatedsample preparation and analysis system 10′ and where similar numberswith primes refer to similar features.

In one embodiment, the system 10 includes a sample preparation system 12for preparing various samples and a sample analysis system 14, whichincludes a suitable analyzer, such as a liquid chromatography massspectrometer (“LCMS”), a gas chromatography mass spectrometer (“GCMS”),a surface desorption/ionizer directly coupled to a mass spectrometer; aliquid chromatography ultra-violet spectrometer (“LC/UV-VIS”), or afluorescence spectrometer, for example, for analyzing the preparedsamples according to selected analyte assays. The sample preparationsystem 12 and the sample analysis system 14 are interconnected in anautomated manner as will be described in detail below and may, in fact,be enclosed within a unitary cover 16.

FIG. 2 is a diagrammatic illustration of various components of thesystem 10. The sample preparation system 12 includes a samplepreparation station 20 and a sample preparation controller 22 thatcontrols selected functions or operations of the sample preparationstation 20. The sample preparation station 20 is configured to receiveone or more specimens 23, to sample the specimens 23 to prepare thesamples for analysis according to a variety of preselected analyteassays, and to transport the prepared samples for analysis to the sampleanalysis system 14. In some embodiments, the sample preparation station20 is configured to prepare the sample such that the prepared sample ischemically compatible with the sample analysis system 14 according tothe selected analyte assay to be performed by the sample analysisstation 14.

Further referring to FIG. 2, in one embodiment the sample analysissystem 14 includes a sample analysis station 24 and a sample analysiscontroller 26 that controls selected functions or operations of thesample analysis station 24. The sample analysis station 24 is configuredto receive the prepared sample from the sample preparation station 20via a transport mechanism described in greater detail below. The sampleanalysis station 24 then analyzes the prepared sample according to aselected analyte assay to obtain a result for that sample. The sampleresult is transmitted to the sample preparation controller 22, which mayvalidate the results. If the result is valid, the result may betransmitted to a laboratory information system 28 (illustrated as, andreferred to hereinafter, as “LIS” 28) via at least one network 30.

It will be readily appreciated that while FIG. 2 seems to indicate thatthe sample preparation station 20 and the sample analysis station 24comprise two opposing sides of the system 10, the systems may encompassthe same area or footprint. Indeed, in accordance with the presentinvention, in some embodiments the sample preparation station 20 and thesample analysis station 24 need not be encompassed within the samehousing or unit.

Turning now to FIGS. 3A-3D, the details of two embodiments of layouts ofthe sample preparation station 20 and the sample analysis stations 24associated with the systems 10, 10′ of FIGS. 1A and 1B are shown andbriefly described below. Additional features are described in detail inInternational Application No. PCT/US0211/58323, entitled “System Layoutfor an Automated System for Sample Preparation and Analysis,” AttorneyDocket No. TFS-13CWO, filed on even date herewith, and incorporatedherein by reference in its entirety. It would be understood that, forthe convenience of discussion, the like reference numerals referring tolike features with primes are included herein, though each is notnecessary provided explicitly. The sample preparation station 20includes a specimen dock 40 having one or more specimen racks 42. Eachspecimen rack 42 includes one or more specimen rack positions capable ofholding a specimen container 45 (see, FIG. 3A). The specimen containers45 are configured to contain the acquired biological or environmentalspecimens, which may be any specimen containing or suspected ofcontaining an analyte of interest. Patient specimens may include blood,serum, plasma, urine, stool, sputum, brochial lavage, nasopharangeallavage, perspiration, tears, extracts of solid tissue, swabs (from allbodily sites, including skin), cerebrospinal fluid, or saliva, forexample. Environmental samples may include, for example, food, water, orenvironmental surface samples. These patient specimens or environmentalsamples may be analyzed for one or more analytes, which may include, butare not limited to, drugs, pro-drugs, metabolites of drugs, metabolitesof normal biochemical activity, peptides, proteins, antibiotics,metabolites of antibiotics, toxins, microorganisms (including bacteria,fungi, and parasites), and infectious agents (including viruses andprions). Further, any of the foregoing samples, alone or in combination,may be suspended in an appropriate media, for example, within a bloodculture or a screening booth. The specimen container 45, itself, mayinclude any suitable labware, such as a vessel, a vial, a test tube, aplate, or any other suitable container known in the art. One or more ofthe specimen racks 42 may be designated, or otherwise labeled (e.g., byplacement of the rack 42 within the sample preparation station 20 orwith a barcode or an RFID antenna), as priority racks 42 a, or STAT, forintroducing specimen containers 45 having urgent specimens.Alternatively, urgent specimens may be introduced into a specimen rack42 and identified as priority or STAT samples by pressing a prioritybutton (not shown) on the instrument or by setting the sample priorityusing a touch screen display 313, which is described in greater detailbelow. Urgent specimens may include, for example, emergency departmentpatient specimens or patient specimens containing toxicants orimmunosuppressants. The manner by which specimens within the priorityrack 42 a are prepared and tested is described in greater detail below.

The specimen dock 40 may be configured to accommodate an on-lineaccession station 47 (see, FIGS. 3C and 3D) for receiving the specimenvessels 45 from an off-line automated laboratory track system (notshown).

The sample preparation station 20 further includes a reagent station 46containing multiple reagent racks 48. Each reagent rack 48 includes oneor more reagent rack positions capable of holding one or more reagentcontainers 52 (see, FIG. 5A) that contain solvents and/or reagents, someof which may be comprised of a volatile liquid. While not necessary, theillustrative embodiment of the specimen racks 42 of the sample dock 40and the reagent racks 48 of the reagent station 46 have similarconstruction. In other embodiments, it may be advantageous to includereagent racks 48 having a different structure as compared to thespecimen racks 42 such that racks 42 containing biological specimens arenot inadvertently inserted into the reagent station 46. In still otherembodiments, reagent racks 48 may include distinct labeling, e.g., abarcode or an RFID antenna, as compared with the specimen racks 42.

The reagent station 46 may include a cooling station 53 coupled to athermostat (not shown) and chiller (not shown) to maintain thetemperature of the reagent station 46 at a constant, cooled temperature,for example, between about 4° C. and about 10° C. This may aid inreducing the loss of reagent through evaporation and thereby extend thelifetime and activity of the reagents contained therein. The reagentcontainer 52 may be similar to the reagent containers that are describedin detail in U.S. Provisional Application No. 61/552,470, entitled“Reagent Bottle, System, Method, and Apparatus for Handling Closure Capsand Like,” naming inventors YY, ZZ, and filed on even date herewith, thedisclosure of which is incorporated herein by reference in its entirety.Alternatively, the reagent container 52 may be similar to the reagentcontainers that are described in detail in U.S. Application PublicationNo. 2008/0093364, the disclosure of which is incorporated herein byreference in its entirety. Briefly, and as shown in FIG. 5A, eachreagent container 52 may include a body 65 and a top wall 66 having sixor more flaps 67 (formed by three or more radially extending incisions)extending radially inward and a flange 68 extending radially outward. Atrest, the flaps 67 are angularly extended inward to form a seal thatreduces evaporation of the reagent contained within the volume of thebody 65. According to one embodiment, the flaps 67 are opened bydirecting a ring-shaped actuator 69, operable by way of one or morerobotics, which forces the ring-shaped actuator 69 downwardly over thetop wall 66 and engages the flange 68. Continued downward movement ofthe ring-shaped actuator 69 lowers the flange 68 and biases the flaps 67upwardly and outwardly, as shown in FIG. 5B.

Returning to FIGS. 3A-3D, various reagents may reside within the reagentstation 46, including all reagents necessary for the plurality of assaytypes that are capable of being performed by the system 10. For example,the reagents may include protein precipitation reagents (e.g.,acetonitrile, methanol, or perchloric acid), cell lysis reagents (e.g.,zinc sulfate, a strong acid, an enzyme digestion with lysozymes,cellulases, proteases, detergents including, without limitation,non-ionic, zwitterionic, anionic, and cationic detergents, proteindigestion reagents (e.g., serine proteases such as trypsin, threonine,cysteine, lysine, arginine, or aspartate proteases, metalloproteases,chymotrypsin, glutamic acid proteases, lys-c, glu-c, and chemotrypsin),internal standards (e.g., stabile isotope labeled analytes, heavyisotope labeled peptides, non-native peptides or analytes, structurallysimilar analogs, chemically similar analogs), antibiotics (formicrobiological antibiotic susceptibility testing, or “AST”), proteinstabilization agents, including buffers, chaotropic agents, ordenaturants, calibration standards, and controls. According to variousembodiments, one or more of the reagents may be pre-mixed to form acombined reagent mixture specific for a particular assay or panel ofassays.

The reagent station 46 may further include an information acquisitiondevice 54 which, for example, may be a bar code reader or an RFIDreceiver. The information acquisition device 54, in turn, may receiveinformation associated with a reagent of the particular reagentcontainer 52 or information associated with the particular reagentcontainer 52 itself. A bar code or RFID antenna is imprinted orpositioned on a reagent container 52. The bar code or RFID antenna maybe configured to provide information associated with the particularreagent or it may contain an identification (such as an identifier) thatis cross-referenced with a Look-Up Table (“LUT”) (not shown) accessibleby the sample preparation controller 22 (FIG. 2) (e.g., on the samplepreparation controller 22 or on the LIS 28 and accessible by the samplepreparation controller 22) and having detailed information regarding thereagent contained therein. The information obtained may be used toidentify and/or monitor a respective reagent container 52 and/or thereagent therein. For example, the information may be used to identifythe reagent within the reagent container 52, identify the location ofthe reagent container 52 within the reagent station 46, identify and/ormonitor the quantity of reagent remaining in the reagent container 52,and/or identify the expiration date of the reagent within the reagentcontainer 52. Though not specifically shown, the information acquisitiondevice 54 may be mounted onto a track system (not shown) that spansbetween the specimen dock 40 and the reagent station 46, and by way ofone or more motors (e.g., a stepper motor or like device) theinformation acquisition device 54 may be translated to a position withinthe specimen dock 40 or the reagent station 46 for receiving a specimenrack 42 or a reagent rack 48. In this way, the information acquisitiondevice 54 may scan the barcode and/or RFID antenna as the specimencontainers 45 and/or reagent containers 52 are loaded into the samplepreparation station 20. Further, it would be understood that while onlyone information acquisition device 54 is shown, additional informationacquisition devices, in like manner or having an alternate structure,may be included in other portions of the system 10 for tracking samplesand the associated tests.

Turning now to an illustrative method of sample preparation, a patientsample (referred to hereinafter as “sample”), or a portion of aparticular specimen contained within a specimen container 45 istransferred to an open-top sample vessel 58 (also known as a reactionvessel, but referred to hereinafter as “vessel” 58) to be prepared foranalysis. Suitable vessels 58 may include, for example, open-topvessels, vessels having a screw-top cap, vessels having integratedflip-top caps, and vessels having tops with piercable septa. Oneexemplary embodiment of a vessel 58 for use with the sample preparationstation 20 is described in detail in U.S. Provisional Application No.61/408,059, entitled “A Reaction Vessel and Apparatus and Method forOpening and Closing a Reaction Vessel,”, naming inventors Nuotio,Siidorov, and Kukkonen, filed on Oct. 29, 2010, and InternationalApplication No. PCT/FI2011/050950, entitled “A Reaction Vessel andApparatus and Method for Opening and Closing a Reaction Vessel,” filedon even date herewith, the disclosures which are incorporated herein byreference in their entirety. Briefly, the vessel 58 of the co-pendingapplication is shown in FIGS. 6A and 6B and includes a body 70 forcontaining one or more of a sample, a reagent, a solvent, and a standard(calibration, control, or internal); and a hinged lid 71 having a guiderod 72 extending upwardly therefrom. At least one rib 73 may be includedexternal to the body 70 for properly aligning the vessel 58 in variousones of the components within the sample preparation station 20.Accordingly, the vessel 58 has an open state (FIG. 6B) to receive one ormore of the sample, reagents, solvents, and standards and a closed state(FIG. 6A) to seal the body 70 and to reduce the evaporation of volatileliquids therefrom. One manner of opening and closing the vessel isdescribed in greater detail below.

Again, returning to FIGS. 3A-3D, the vessels 58 may be stored within,and introduced from, a storage station 59 (FIG. 3D) of the samplepreparation station 20. Within the storage station 59, the vessels 58may reside in plates 57 or other appropriate mass storage containers. Asvarious ones of the vessels 58 are transferred and periodically leavingempty plates 57, the plates 57 may be discarded through a waste chute 55from the sample preparation station 20.

When a specimen 23 (FIG. 2) is sampled, one or more vessels 58 aretransferred to a sampling station 56 from the storage station 59 (FIG.3D) by way of a transport assembly 60. The transport assembly 60 mayinclude a robot assembly operating on one or more tracks 50 andconfigured to move in at least one of an x-y direction, an x-y-zdirection, or a rotary direction. An exemplary track system andassociated transport bases are described in detail in U.S. Pat. No.6,520,313, entitled “Arrangement and Method for Handling Test Tubes in aLaboratory,” naming inventors Kaarakainen, Korhonen, Makela, and whichis hereby incorporated herein by reference in its entirety.

While not shown, the transport assembly 60 may further include agripper, or other like device, to capture and release the vessel 58 or atransport handle 63 (FIG. 3B) associated with a vessel rack 84 (FIG. 3B)to simultaneously transport two or more vessels 58 within the system 10.An exemplary gripper for use on the transport assembly 60 is describedin detail in U.S. Provisional Application No. 61/408,051, entitled“Method and Assembly for Transporting Single and Multiple ReactionVessels,” naming inventor Nuotio, filed on Oct. 29, 2010, andInternational Application No. PCT/FI2011/050949, entitled “Method andAssembly for Transporting Single and Multiple Reaction Vessels,” filedon even date herewith, the disclosures of which are incorporated hereinby reference in their entirety.

In another embodiment, not shown, the transport assembly 60 may includea robot assembly configured to move in at least one of an x-y direction,an x-y-z direction, or a rotary direction and which may include anautomated liquid handler. According to this embodiment, the automatedliquid handler may aspirate and dispense a volume of a liquid betweentwo or more vessels 58 within the system 10 to transport the liquidbetween two or more stations 20, 24, 40, 46, 47 within the system 10.

In still other embodiments, the transport assembly 60 may furtherinclude carousels, i.e., a circular revolving disc, or autosamplershaving multiple vessel positions therein to provide transport functionand allow for a temporary, intermediate vessel storage function. Inother embodiments, the transport assembly 60 may further include aninformation acquisition device (not shown). This information acquisitiondevice may operate in a manner similar to the information acquisitiondevice 54 and be used to identify the vessels 58 as they are movedthroughout the sample preparation station 20.

In the illustrated embodiment, the sampling station 56 includes arotatable table 44 that rotates each vessel 58 between at least twopositions. In one position, the vessel 58 may be received from thetransport assembly 60. In another position, the vessel 58 is positionedto receive a portion of the specimen 23 (FIG. 2) via a sample pipetteassembly 62 (FIG. 4A). The rotatable table 44 may include a vessel capopening and closing device 64 (FIG. 3B) for opening and closing thehinged lid 71 (FIG. 6A) of the vessel 58 as the vessel 58 is transportedbetween the first and second positions. Accordingly, the hinged lid 71(FIG. 6A) may be in the closed position (FIG. 6A) when the vessel 58 isdelivered to the first position of the sampling station 56. Rotation ofthe rotatable table 44 moves the vessel 58 to the second position andcauses the guide rod 72 (FIG. 6A) to engage the opening and closingdevice 64, thereby opening the hinged lid 71 (FIG. 6B). One suchrotatable table with opening and closing device is described inaforementioned U.S. Provisional Application No. 61/408,059, entitled “AReaction Vessel and Apparatus and Method of Opening and Closing aReaction Vessel,” filed on Oct. 29, 2010, and International ApplicationNo. PCT/FI2011/050950, entitled “A Reaction Vessel and Apparatus andMethod for Opening and Closing a Reaction Vessel,” filed on even dateherewith, and the disclosures of which are incorporated herein byreference in their entireties.

The sample pipette assembly 62 (FIG. 4A) may include a pipette shaft 74(FIG. 4A) that is movable, for example via a robotic device, in one ormore of the x-y-z directions and between two or more of the specimendock 40, the reagent station 46, and the sampling station 56. Thepipette shaft 74 (FIG. 4A) may be constructed with a single tipconstruction that is washed between aspirations at a pipette washstation 76 or, alternatively, may be adapted to receive a disposable tip(not shown) that is then ejected before acquiring a new disposable tipto aspirate a new sample. In the former embodiment, after a sample isdispensed to the vessel 58, the single tip is washed, one or more times,by an appropriate solvent solution, such as by multiple aspirations anddispensing of the solvent. In the latter embodiment, the samplepreparation station 20 may include a tip storage station 75 for storageand supplying disposable tips from one or more disposable tip racks 77.After the sample is dispensed to the vessel 58 in this embodiment, thedisposable tip is ejected from the pipette shaft into a disposable tipwaste chute 78, which is coupled to a larger waste storage container(107).

The sample pipette assembly 62 (FIG. 4A) may aspirate an aliquot of thespecimen 23 (FIG. 2) from the specimen container 45 from within thespecimen dock 40 and dispense the aliquot of the specimen 23 (FIG. 2)into the vessel 58 within the sampling station 56. Additionally, oralternatively, the sample pipette assembly 62 (FIG. 4A) may aspirate analiquot of a desired reagent from one of the reagent containers 52within the reagent station 46 and dispense the aliquot of the desiredreagent into the vessel 58 within the sampling station 56, which may ormay not previously include the sample, i.e., the aliquot of the specimen23 (FIG. 2).

In some embodiments, it may be necessary to mix the specimen 23 (FIG. 2)prior to aspirating. For example, blood specimens may partition overtime, i.e., separation of blood cells (erythrocytes, leukocytes, etc.)from the plasma. Some drugs are distributed unequally between the bloodcells and the plasma (for example, with 40-50% in erythrocytes, 10-20%in leukocytes, and 30-40% in the plasma). These distributions may bedependent on temperature, hematocrit, and metabolite concentration.Thus, in order to properly measure the particular drug, or otheranalyte, concentration, a proper sampling of the whole blood must beacquired. One method of mixing the specimen may occur by aspirating anddispensing the specimen 23 (FIG. 2) a number of times (for example, 13times) with the sample pipette assembly 62 (FIG. 4A). The number ofaspirations may depend on at least the volume of the specimen container45, the aspiration volume, and the dispensing “speed.” One method ofmixing the specimen 23 (FIG. 2) using aspiration and dispensing isdescribed in detail in U.S. Provisional Application No. 61/552,472,entitled “Method for Treating a Sample,” filed on even date therewith,and the disclosure of which is incorporated herein by reference in itsentirety.

In other embodiments, the pipette shaft 74 (FIG. 4A) may shake (moverapidly in at least one dimension) or the sample may be first directedto mixing station (not shown) that is separate from the sampling station56 to gently mix the specimen 23 (FIG. 2) prior to sampling.

According to still another embodiment, the sample is selected from aculture plate using a commercially-available colony picker instrument,for example, the PICKOLO (Tecan Group, Ltd., Männedorf, Switzerland) orthe QPIX (Genetix, now part of Molecular Devices, San Jose, Calif.). Thecolony picker is capable of collecting an aliquot of the specimen 23(FIG. 2) from a colony, optionally, a pre-selected or pre-designatedcolony, on a culture plate and depositing the sample into the vessel 58.The colony-containing vessel may then be mixed, as described above, tolyse the cells and denature the proteins in order to stabilize thesample for later microbial analysis.

Following receipt of the aliquot(s) of the specimen 23 (FIG. 2) and/orreagent (which will now be referred to hereinafter for convenience asthe “sample”) the hinged lid 71 (FIG. 6A) is closed via a separaterobotic component or the opening and closing device 64. Closing thehinged lid 71 prevents loss of the sample through evaporation. Moreover,because one or more of the liquids dispensed into the vessel 58 may bevolatile, sealing of the vessel 58 may be used to prevent evaporation ofthe one or more volatile liquids and preserve the intended concentrationof the sample.

The sample within the vessel 58 is transferred via the transportassembly 60 from the sampling station 56 to a secondary processingstation 80. The secondary processing station 80 includes, for example,one or more of a mixing station 82, an incubation station (not shown),and a matrix interference removal station (illustrated as a centrifuge88). According to one embodiment, each of the mixing station 82 and thematrix interference removal station is capable of accepting eithervessels 58 or a vessel rack 84 such that two or more vessels 58 may beprocessed simultaneously. However, the use of the vessel racks 84 is notrequired.

The mixing station 82, if included in the secondary processing station80, may include a shaker, a vortex mixer, or another apparatus capableof accelerating the mixing of the sample within the vessel 58. As shown,the mixing station 82 may be configured to accommodate sixteen vessels58 (FIG. 3A) or twelve individual vessels 58 via two vessel racks 84(FIG. 3B).

The incubation station, if included, may also be incorporated in eitherof an on-line or off-line configuration. The incubation station isconfigured to heat a sample, with or without additional reagents, at anelevated temperature (by way of example, at a specified temperatureranging from about 27° C. to about 70° C., including, particularly, 37°C., 50° C., or 70° C.) for a predetermined duration of time and based ona selected assay. For example, the incubation temperature and durationfor a particular sample type and/or selected assay type may bedetermined by incubating a panel of the same sample and assay types at arange of temperatures and/or a range of times. Following incubation,each sample in the panel is analyzed with a control standard and thedigestion efficiency of the incubation is calculated. The temperatureand time producing the optimal digestion efficiency may then be selectedas a preset or previously determined incubation temperature and time forthat sample and assay type.

The matrix interference removal station, if included within thesecondary processing station 80, may be incorporated in either of anon-line or off-line configuration (e.g., the on-line configuration beinga configuration in which the sample moves between one or more stationsof the sample preparation station 20 through fluidic connections withoutbeing contained in a vessel 58, the off-line configuration being aconfiguration in which the sample is transported within a vessel 58between stations of the sample preparation station 20). In embodimentsthat include an on-line matrix interference removal station, theanalyte-containing prepared sample may flow directly from the matrixinterference removal station to the next station, such as throughtubing. This second station may include, for example, a second matrixinterference removal station (not shown). In embodiments that include anoff-line matrix interference removal station, the analyte-containingprepared sample is collected from the matrix interference removalstation and placed into a vessel 58 if not already contained in a vessel58.

The matrix interference removal station is operable to separate one ormore of residual proteins, phospholipids, salts, metabolites,carbohydrates, nucleic acids, and/or other substances that may otherwiseinterfere with subsequent processing or analysis of the desired analytesand prior to transferring the now prepared sample to the sample analysisstation 24. In some embodiments, the matrix interference removal stationseparates contaminants from the analyte-containing prepared sample, ormore simply, the “prepared sample” (for example, by separatingprecipitated solids from a resulting supernatant liquid, wherein thesupernatant liquid forms the prepared sample). The matrix interferenceremoval station may include, for example, one or more of a phaseseparation station (not shown), a centrifuge (illustrated as referencenumber 88 in FIG. 3A and reference number 88′ in FIG. 3B), a sonicator(not shown), a heating station (not shown), a flash freeze station (notshown), an affinity purification station (not shown), or a filtrationstation (not shown). Each embodiment of the matrix interference removalstation may be configured to accommodate one or more vessels 58 or oneor more vessel racks 84, or the contents of one or more vessels 58, asappropriate.

One of ordinary skill in the art will readily appreciate thatincorporation of the centrifuge 88 into the housing 16 with theanalytical instrumentation of the system 10 may cause undesirableinterference with those analytical instruments. In this integralconfiguration example, the ability of the analytical instruments toperform with a particular reliability may be compromised. This may bedue to, at least in part, the high rotational speed required to drawdown the precipitating solids from the supernatant liquid. Therefore, itmay be necessary for embodiments of the system 10 including anintegrated centrifuge 88 to further include features that reducetransmission of vibrations thereof to other components of the system 10.Moreover, because of the desire to reduce the overall footprint of thesystem 10, the overall size of the centrifuge 88 may be reduced and/orconfigured to be a standalone centrifuge 88 that is not integral withother components of the system 10, but yet accessible by the transportassembly 60.

The matrix interference removal station may include an affinityextraction or purification station, for example, an immunoaffinityextraction or purification system. An exemplary immunoaffinity systemmay use a Mass Spectrometry Immunoassay (“MSIA”) antibody enrichedsubstrate. One suitable, commercially-available MSIA substrate includesthose from Intrinsic Bioprobes Inc. (Tempe, Ariz.) and that aredescribed in U.S. Pat. No. 6,783,672, the disclosure of which isincorporated herein by reference in its entirety. More specifically,MSIA substrates may include monolith substrates formed of glass,crystal, or metal having attached antibodies with a specific affinityfor the analyte of interest. MSIA substrates may be developed withantibodies specific to the analyte to be extracted. Exemplary analytesinclude, but are not limited to, small molecules, small moleculevariants, proteins, protein fragments, and protein variants. Duringanalyte extraction, the sample is aspirated into the MSIA pipette tip orpassed through the MSIA column so that the analyte of interest forms anaffinity association with the attached antibody. Following associationof the analyte with the antibody, the remaining matrix portion of thesample is dispensed from the MSIA tip or column. The analyte of interestis subsequently eluted from the antibodies into an appropriate mediathat is configured to release the analyte from the antibody for furtheranalysis. One exemplary MSIA method is described in greater detail inU.S. Pat. No. 6,974,704, the disclosure of which is incorporated hereinby reference in its entirety.

Though not specifically shown, the matrix interference removal stationmay, in yet other embodiments, include additional techniques known inthe art of chemical separation, such as liquid-liquid extraction, solidphase supported liquid extraction, random access media columnextraction, monolithic column extraction, dialysis extraction,dispersive solid phase extraction, solid phase micro-extraction,immunoaffinity extraction, and size exclusion using membrane filterswith gravity, vacuum, or centrifugation. Many of these techniques may bepracticed off-line or on-line, as appropriate, if fluid connections arecreated between subsequent steps of the method. Additionally, many ofthese techniques may be practiced in a variety of formats including, forexample, using a column or cartridge format, using a pipette tip format,using a magnetic bead format, or using a plate or chip format.

In still yet another embodiment, the matrix interference removal stationmay include two or more matrix interference removal methods in series.According to this embodiment, the first matrix interference removalstation, for example a phase separation station, removes precipitatedproteins while the second matrix interference removal station, forexample a solid phase extraction station, removes additional residualproteins, phospholipids, and salts from the sample prior to analysis.Additional examples of combinations of matrix interference removaltechniques include, but are not limited to, solid phase extractionfollowed by liquid-liquid extraction, phase separation followed by sizeexclusion affinity liquid chromatography, solid phase extractionfollowed by size exclusion affinity liquid chromatography, andimmunoaffinity extraction prior to or following any of theaforementioned methods.

Embodiments of the system 10 that include a filtration station may inturn include a vacuum and/or positive pressure filtration system (notshown) for passing the sample through a filter membrane (not shown) forcollecting and/or removing precipitated proteins or other solidparticles from the sample. The vacuum and/or positive pressure systemmay, or may not, include a filter (for example, a speedvac), to dry downthe sample in order to remove a liquid, for example a solvent, from thesample. Alternatively, the filtration station may include a HighPerformance Liquid Chromatography (“HPLC”) system (not shown) or anUltra-High Performance Liquid Chromatography (“UHPLC”) system containingat least one LC column having suitable stationary and mobile phases toremove matrix interference. In some embodiments, the filtration stationmay in actuality include two or more LC columns arranged on-line and inseries (often referred to as two-dimensional LC or multi-dimensionalLC). Examples of suitable LC columns include a size exclusionchromatography, high turbulence chromatography, a reversed-phasechromatography, ion-exchange chromatography, bio-affinitychromatography, or other as known in the art.

Embodiments of the system 10 that include a phase separation componentmay include an on- or off-line solid phase extraction station (notshown) having a vacuum and/or positive pressure source (not shown) toassist in moving the sample through a solid phase extraction matrix. Thesolid phase extraction matrix, in turn, may include one or more suitableporous stationary phase material layers. According to one embodiment,the solid phase extraction matrix (not shown) further includes one ormore filters arranged on one or both sides of the porous stationaryphase material. The solid phase extraction matrix may be arranged, forexample, within a column, cartridge, a pipette tip, or in a plate orchip format.

After the sample has passed through the secondary processing station 80,the prepared sample is transported via the transport assembly 60 to ananalysis staging station 90. The analysis staging station 90 includestwo or more vessel positions 94 (FIG. 3A) or two or more vessel rackpositions 94′ (FIG. 3B) for accepting vessels 58 or vessel racks 84,respectively. In the particular illustrative examples, the analysisstaging stations 90, 90′ accommodate about one hundred thirty-twovessels 58 (FIG. 3A) or twenty-four vessel racks 84 (FIG. 3B) that are,in turn, capable of accepting up to about six vessels 58 each, for atotal of one hundred forty-four vessels 58. Each vessel position 94 maybe stationary within the analysis staging station 90 such that once anindividual vessel 84 is placed within a vessel position 94 of theanalysis staging station 90, its position does not change but fortransfer by the transport assembly 60.

While not specifically shown, the analysis staging station 90 mayinclude a cooling system to maintain the temperature of the analysisstaging station 90 at a constant controlled temperature, for example, atemperature of about 4° C. to about 10° C. In this way, and along withthe hinged lid 71 (FIG. 6A) described previously, the rate ofdegradation or evaporation of the prepared sample is further reducedwhile the prepared sample is awaiting analysis.

Alternatively, the analysis staging station 90 may include a heatingsystem, or an incubation station, as described above, to maintain thetemperature of the analysis staging station 90 at a constant controlledtemperature for incubation, for example, a temperature ranging fromabout 23° C. to about 70° C. Some specimen types, particularlymicrobiological specimens, may require extended incubation of theotherwise prepared sample prior to analysis. Following completion of theincubation time designated for the specific sample, the prepared andincubated sample may be selected for analysis according to the analysisselection criteria described in detail below.

When a particular prepared sample is selected for analysis (the detailsof the selection criteria being described in detail below), the vessel58 containing the prepared sample is transferred via the transportassembly 60 from the analysis staging station 90 to an injector station92. The injector station 92 may include an injector pipette assembly 96(FIG. 4A) to transfer an aliquot of the prepared sample from the vessel58 to the sample analysis station 24. The injector pipette assembly 96(FIG. 4A) includes a pipette shaft 97 (FIG. 4A) that may be constructedin a manner that is similar to the sample pipette assembly 62 (FIG. 4A)that was described in detail above.

According to that embodiment, and as shown in FIGS. 3A-3D and FIG. 4A, awash station 98 having a solvent supply 99 fluidically coupled theretois provided to wash the pipette shaft 97 between aspiration-dispensingas necessary. In another embodiment, not particularly shown herein, thepipette shaft 97 may be shaped to receive a disposable tip, which may beprovided in addition to or in alternative to the wash station 98.

The injector station 92 may include a rotatable table 102 having astructure that is similar to the sampling station 56 and may include avessel cap opening and closing device 103 for opening and closing thehinged lid 71 (FIG. 6A) of the vessel 58 as it is transported to a readyposition 100. Following aspiration of an aliquot of the prepared sampleby the injector pipette assembly 96, the vessel 58 may be rotated awayfrom the ready position 100, thereby closing the hinged lid 71 (FIG.6A), and preparing the vessel 58 for receipt by the transport assembly60 for transport and release to a storage facility (not shown) or awaste chute 106 leading to the vessel waste storage 107. Disposablepipette tips may also be ejected from the pipette shaft 97 into thewaste chute 106.

As described in detail above, a sample of a specimen 23 (FIG. 2) isprepared at the sample preparation station 20 before that preparedsample is moved to the sample analysis station 24. As such, at leastsome of the movable portions of the sample preparation station 20,including the sampling station 56, the transport assembly 60, therotatable tables 44, 102, the injector station 92, and the injectorpipette assembly 96, acting individually or in concert, may comprise atransport mechanism to transport the prepared sample from the samplepreparation system 12 to the sample analysis system 14. One havingordinary skill in the art will appreciate that alternative embodimentsof a transport mechanism to transport a prepared sample from a samplepreparation system 12 to a sample analysis system 14 may be used withoutdeparting from the scope of embodiments of the invention. In theexemplary embodiment, the transport mechanism may comprise the injectorpipette assembly 96, which removes an aliquot of the prepared sample fordispensing to the sample analysis station 24.

Turning now to the details of the sample analysis station 24, and inparticular to FIGS. 3A-3D and 4B, one embodiment of the sample analysisstation 24 may be an LCMS system having a liquid chromatography station110 and a mass spectrometer station 112. The liquid chromatographystation 110 (referred to hereinafter as “LC station” 110) may includeone, two, or more injection ports 104 a, 104 b for accepting the aliquotof the prepared sample from the injector pipette assembly 96 foranalysis. The injection ports 104 a, 104 b may be connected on-line toone or more chromatography columns (e.g., a preparatory column 114 andan analytical column 116) for separation of the prepared sample intoanalytes of interest eluting at one or more elution times and aplurality of ancillary or waste eluents. As shown in the illustrativeembodiments, the LC station 110 includes two separation channels, i.e.,LC channels 118 a, 118 b (a third LC channel 118 c shown in phantom).Each LC channel 118 a, 118 b, 118 c includes one preparatory column 114and one analytical column 116, arranged in series. The preparatorycolumn 114, according to some embodiments, may be a size exclusionaffinity liquid chromatography column used for, in essence, matrixinterference removal. The analytical column 116 may be a reversed-phaseLC column for analyte isolation. Exemplary embodiments include a CycloneP 0.5×50 mm TURBOFLOW size exclusion affinity liquid chromatographycolumn (Thermo Fisher Scientific, Inc., Waltham, Mass.), described indetail in U.S. Pat. Nos. 5,772,874; 5,919,368; and 6,149,816, thedisclosures of which are all of which are hereby incorporated herein byreference in their entireties, and a Hypersil GOLD PFP 2.1×50 mm, 1.9μUHPLC analytical column (Thermo Fisher Scientific, Inc., Waltham,Mass.). The columns, according to additional embodiments, may becapillary columns (having an internal diameter of approximately 300 μm),nano columns (having an internal diameter ranging from about 74 μm toabout 100 μm), available in packed tip formats, standard packed formats,and biphasic columns for two-dimensional work, for example.

Briefly stated, the TURBOFLOW turbulent flow liquid chromatographyapparatus includes a chromatography column that is formed as asubstantially uniformly distributed multiplicity of rigid, solid, porousparticles (“stationary phase”) having substantially uniform meancross-section dimensions and a plurality of pores within each particle.The particles are selected from a range of various sizes and shapes andare held together in a column by pressure, sintering, or the like sothat interstitial channels having a total interstitial volume of notless than some designated percentage of the total volume of the column.The surfaces of the particles, including the inner surfaces of the poresin the particles, are chromatographically active, as by being coatedwith chromatographic stationary phase layers. In operation, theturbulent flow of the mobile phase through the TURBOFLOW column causeslarger molecules to elute more rapidly than smaller molecules, thelatter of which filter through the pores of the particles comprising thestationary phase. Further differentiation in elution times of variousmolecules is based, at least in part, in the molecules affinity to thecoating of the particles. By adjusting the mobile phase (e.g., the pH,relative concentration of aqueous and organic solvents, and so forth)the affinity of one or more molecules is altered and the variouscomponent molecules isolated or separated.

In other embodiments, the preparatory column 114 may be a conventionalsize exclusion column or any other liquid chromatography column that maybe utilized as a matrix interference removal device.

Each of the two LC channels 118 a, 118 b is associated upstream with arespective injector port 104 a, 104 b and associated downstream with asingle mass spectrometer 120 of the mass spectrometer station 112 in amanner that enables multiplexing or staggered sample introduction intothe mass spectrometer 120 as described in detail below.

The selection of the appropriate preparatory and analytical columns 114,116 may be based, at least in part, on the ability of an LC channel 118a, 118 b to provide a range of retention times for analytes of interestversus the eluent. Conventionally, the preparatory and analyticalcolumns 114, 116 are matched for a particular analyte from a particulartype of sample; however, the matching of columns 114, 116 may vary fromone assay type to another. More specifically, each desired analyte willhave a ratio of concentration distribution between two immisciblesolvents (e.g., octanol and water a solvent from supply 129) atequilibrium, which is referred to as the partition coefficient. Saidanother way, the partition coefficient is a measure of a desiredanalyte's hydrophilicity (“water loving”) or hydrophobicity (“waterfearing”). Often the partition coefficient is reported as the logarithmof the partition coefficient, referred simply as “logP.” It would bereadily appreciated that because logP is logarithmic in nature, eachunit represents a difference in hydrophobicity by factor of 10. Thus,two analytes having similar logP values may have similarhydrophobicities (i.e., chemistry) and may be easily separated by thesame mobile and stationary phases; two analytes having different logPvalues tend to result in vastly different retention characteristics onthe same LC system and may require quite different mobile and/orstationary phases to achieve a desired chromatographic separation. As aconsequence, analytes are conventionally grouped together according tosimilar logP values and run in batch modes using the same mobile phaseand mass spectrometer settings.

In a conventional system, when a new prepared sample is to undergotesting in accordance with an assay type that is different from theassay type for which the system is presently configured, one or more ofthe columns must be exchanged. Exchanging a column requires a user toassemble a compression fitting and ferrule onto the end of afluid-carrying length of tubing. The compression fitting and tubing isinserted into a column end fitting of the column and tightened with twoproperly sized wrenches. While the user must ensure that the junction isnot under tightened (leading to a leak), great care must also be takento not over tighten the joint (leading to galling, breakage, or tubecollapse). Further, repeated exchange of columns may lead to materialfatigue and increased likelihood of galling, breakage, or tube collapse.

Alternatively, one or more universal sets of columns may be used wherethe universal set of columns has an ability to process and isolate avariety of analytes of interest, including and up to the number of assaytypes processed by the system 10. In this way, the assay menu includinga plurality of analytes may require only one or a few select universalsets of columns, thus greatly simplifying the user interface and theuser interaction with the system 10. While the universal sets may bedescribed as a preparatory column with an analytical column 114, 116, itwill be understood that the sets may comprise any number of columns.Embodiments of such universal sets of columns are described in U.S.Provisional Patent Application No. 61/408,266, entitled “LC-MSConfiguration for Purification and Detection of Analytes Having a BroadRange of Hydrophobicities,” naming inventors Herman, DeWitte, Jardine,and Argot, filed on Oct. 29, 2010, and International Application No.PCT/US11/58430, entitled “LC-MS Configuration for Purification andDetection of Analytes Having a Broad Range of Hydrophobicities,” filedon even date herewith, the disclosures of which are incorporated hereinby reference in their entireties.

Briefly, each universal column isolates, or purifies, a broad range ofdesired analytes based on, for example, the relative hydrophobicities ofthe analytes using one set of LC columns and one set of mobile phasebuffers per set. Isolation may be further dependent on mobile phase flowrate, relative ratios of one mobile phase buffer to another or others,temperature, and so forth. Instead of manually changing columns andmobile phases as described above, the universal method allows LCMSpurification and analysis of a broad range of analytes, having a broadrange of logP values, to be accomplished using one set of LC columns andone set of mobile phase buffers by selecting LC system parameters (e.g.,flow rate, ratios of one buffer to another, temperature, and the like)and MS system parameters (e.g., ionization voltage, desolvationtemperature, and the like) that are particular to a range of logP valuesor different classes of analytes.

It will be understood that the universal set of columns need not belimited to single set of columns, but may instead include two or moresets that are specifically directed to a given range of logP values,i.e., class of analytes. Then, and in accordance with the methodsdescribed in greater detail below, the two or more LC channels 118 a,118 b may be multiplexed to greatly enhance the efficiency of the system10. Each of the two or more LC channels 118 a, 118 b may be assigned toa particular universal set of columns for a particular class ofanalytes. Further, two or more of the LC channels 118 a, 118 b may beassociated with similar or overlapping ranges of logP values and thuseither of the channels may be appropriate used to elute certainanalytes.

Indeed, in yet other embodiments, the set of universal columns may bepackaged within a unitary cartridge, or housing, for ease of exchangingcolumns as necessary, and as provided in greater detail in InternationalApplication No. PCT/US2011/58229, and U.S. Provisional Application No.61/408,044, both entitled “Modular Multiple-Column ChromatographyCartridge,” naming inventor Brann, filed on Oct. 28, 2011, and Oct. 29,2010, respectively, the disclosure of which are incorporated herein byreference in their entireties. One such dual-column chromatographycartridge 113 is shown in FIG. 7A according to a longitudinalarrangement, with an alternate arrangement shown in FIG. 7C according toa lateral arrangement. The cartridge 113 includes a housing 115 havingtwo chromatography columns—a first column 114 and a second column 116—atleast partially contained therein. Preferably, but not necessarily, thecolumns 114, 116 are affixed to the housing 115. The two columns 114,116 in each cartridge 113 may be matched for purposes of conductingchromatographic separations of a specific analyte (or analytes). Forinstance, the first column 114 may comprise a cleanup column such as theTURBOFLOW, described above, or an HPLC column while the second column116 may comprise an analytical column. The first column 114 comprisestwo column end fittings 108 a with one such end fitting 108 a at eachend of the column 114. Likewise, the second column 116 comprises an endfitting 108 b at each end of the column 116. As is known, column endfittings 108 a, 108 b are attachment points for fluidic connections toexternal tubing 121. Accordingly, one or more connectors 117, 119 areemployed in order to connect the two end fittings 108 a of the firstcolumn 114 to fluid tubing 121 as well as to connect the two endfittings 108 b of the second column 116 to fluid tubing 121. The tubing121 may fluidically couple one or both ends of the first column 114 toat least one valve (two valves 126 a, 126 b are shown) so as to directflow in either direction through the first column 114, depending onwhether the column 114 is being loaded or flushed. The tubing 121 isalso fluidically coupled to the second column 116 with for providingfluid to the second column from the second valve 126 b and outputtingeluting analytes to another valve 133. It would be readily appreciatedthat the tubing 121 may be coupled to the column ends of the secondcolumn 116 in an opposite sense to that shown in FIG. 7A such that fluidflow through the second column 116 would be in the opposite direction tothat shown.

In some embodiments, the cartridge 113 may include a passive indicator123 capable of being read by a barcode reader (not shown) or otherapparatus of the system 10 (FIG. 1A). The combination of a passiveindicator 123 and a reader may enable system software to be able toautomatically verify that the columns 114, 116 within a selected andinserted cartridge 113 are appropriate for an analysis protocolcurrently being conducted by the system 10 (FIG. 10).

The cartridge 113 may further, or alternatively, include variouselectronic control, sensing, data storage or logic components togetherwith associated external electronic connectors. For example, thecartridge 113 may include one or more heaters 125 a in intimate contactwith one or more of the contained columns (though only association withthe first column 114 is shown) so as to control temperature during achromatographic procedure. The heater 125 a may be used, for instance,to increase temperature so as to release a sample fraction previouslyretained or concentrated on a stationary phase within the first column114 such that the sample fraction may be transferred to the secondcolumn 116. The heater 125 a may comprise any of a several heatingdevices such as a coiled resistance wire or commercially-availableheating tape, for example. In that regard, the cartridge 113 may includeone or more temperature sensors 127. Such sensors 127, if present, maywork in conjunction with any on-board heaters 125 a as part of atemperature control loop to control the temperature of the one or morecolumns 114, 116. The control logic may be implemented in software of acontroller, generally, the sample preparation controller 22 (FIG. 2),specifically, or, alternatively, may be implemented in firmware of anon-board circuitry module 111 which may comprise electronic memory orcontroller logic. In addition, or alternative, the circuitry module 111may be used to actively record computer-readable data or otherinformation pertaining to the module, including module historyinformation, wherein such information is downloadable by ortransferrable to external apparatus (such as the sample preparationcontroller 22 of FIG. 2) through a standard interface 109, such as a USBport. Sill other electrical or electronic connectors 105 may be employedto provide power to the heater 125 a, to read the sensor(s) 127, etc.

Depending upon the rate of fluid flow, the configuration illustrated inFIG. 7A may not allow sufficient time for the fluid within the one ormore columns 114, 116 to achieve the desired temperature. Therefore, asecond heater 125 b may additionally, or alternatively, included asshown in FIG. 7B. The second heater 125 b is configured such that thefluid is in contact with a heated length of the tubing 121 for asufficient time so as to achieve the desired temperature within thefirst column 114. The second heater 125 b may comprise any of a severalheating devices described above.

FIG. 7D illustrates a perspective view of a cartridge 113′ in accordancewith another embodiment of the present invention. In many situations, itmay be desirable to fabricate a cartridge 113′ so as to allow the userto simply, with one motion, insert the cartridge 113′ into a holder orcradle 1118 (FIG. 8) such that electrical connections to the cartridge113′ are made at the same time that correct cartridge positioning isachieved. Accordingly, the cartridge 113′ may comprise a singleelectrical connector port 131 that replaces and provides thefunctionality of both the standard interface port 109 and the separateelectrical connector 105 shown in FIGS. 7A and 7B. The connector port131 is shown on the underside of the cartridge 113′, in this example,since it may be advantageous for the user to insert the cartridge 113′by applying a downwardly-directed force into a cradle 1118, such as theone described in greater detail below in reference to FIG. 8. The singledownward motion will then both align the cartridge 113′ and cause theconnector port 131 to make contact with a mating connector in, forinstance, a base plate 1222. Other connector port and insertionconfigurations are also possible and are not limited to the specificillustrative embodiments shown herein.

FIG. 8 illustrates a cartridge 113′ mounted on cartridge support members1220, which are mounted on the base plate 1222. A plurality of connectorapparatii 1224 a, 1224 b, 1224 c, 1224 d are also mounted on the baseplate 1222. Grooves 1225 in each of first and second connector apparatii1224 a, 1224 b connect the inlet and outlet connectors 117′ of the firstcolumn 114 (FIG. 7A) of the cartridge 113′ to associate the fluid tubing121 via an end fitting 1227; similar grooves 1225 in each of third andfourth connector apparatii 1224 c and 1224 d provide similar functionswith respect to the second column 116 (FIG. 7A) of the cartridge 113′.In this example, electronic or other electrical connections to thecartridge 113′ may be facilitated by a connector (not shown) mounted tothe base plate 1222 and positioned beneath the cartridge 113′.

Each connector apparatus 1224 a, 1224 b, 1224 c, 1224 d furthercomprises a body 1226 and a piston 1228. The body 1226 includes an openbore 1230. The piston 1228 is capable of being slidably inserted atleast partially into the bore 1230 of the body 1226 and is also capableof being at least partially retracted from the bore 1230. Preferably, aportion of the bore 1230 comprises a shape that mates with the piston1228 such that the piston 1228 is capable of being slidably insertedinto the bore 1230. A bushing or other bearing 1234 may be providedwithin the portion of the bore 1230 that receives the piston 1228 so asto provide a smooth sliding surface for the reciprocating movement ofthe piston 1228. The reciprocating movement of the piston 1228 withinthe body 1226 may be controlled manually by a user by means of a pushingand latching mechanism 1236 (hereafter “locking mechanism” 1236). Asshown, the locking mechanism 1236 may comprise a hand operated lever1238 and a coupling bar 1240 such that the coupling bar 1240 ismechanically engaged to the lever 1238 by means of a first pivot pin1242 about which an end of the coupling bar 1240 is free to rotate. Asecond pivot pin (not shown) similarly provides mechanical engagementbetween the opposite end of the coupling bar 1240 and the piston 1228 sothat rotational motion of the lever 1238 is converted into translationalmotion of the piston 1228. With sufficient translational motion, thepiston 1228 engages and forms fluid communication with the outletconnectors 117, 118.

While FIGS. 4B and 7A-7B are illustrative of embodiments of liquidchromatography plumbing, one of ordinary skill in the art will readilyappreciate that additional plumbing arrangements may be used. Forexample, the preparatory column 114 and the analytical column 116 neednot be fluidically coupled in series. Instead, more complex plumbingarrangements may be used where the LC channels 118 a, 118 b are notisolated, one or more columns 114, 116 may be by-passed as necessary,and so forth. Exemplary plumbing arrangements are further described inC. Chassaing and Robins, S., “Turbulent Flow Chromatography: an EvolvingSolution for Bioanalysis.” CHROMAT. TODAY 2009; 9:20-24; L. Du, et al.“High Turbulence Liquid Chromatography Online Extraction and Tandem MassSpectrometry for the Simultaneous Determination of SuberoylanilideHydroxamic Acid and its Two Metabolites in Human Serum.” RAPID COMM.MASS SPECT. 2005; 19:1779-1787; and T. Edge, “Chapter 4: Turbulent FlowChromatography in Bioanalysis.” In: I. D. Wilson, ed. HANDBOOK OFANALYTICAL SEPARATIONS. Elsevier Science B.V. 2003; Vol 4:91-128; eachreference is incorporated herein by reference in its entirety.

Referring still to FIG. 4B, each LC channel 118 a, 118 b may be furtherassociated with at least one pump 124 and at least one valve 126 a, 126b to control the flow of the mobile phases and the prepared samplethrough the sample analysis station 24.

While any one of a number of various pumps, including, for example,reciprocating, dual piston, or peristaltic pumps, these conventionalpumps require multiple strokes per sample injection. By contrast,syringe pumps may be considered to be preferred to inject the preparedsample and the mobile phases from a mobile phase container 122 into andthrough the columns 114, 116 and interconnected tubing lines because ofthe reduced volume of mobile phase and reduced dead volume required foreach analysis and the reduced wear on the LC station 110 overall. Witheach stroke of the piston of a conventional pump, a slight pressuredifferential may be created within the LC channel 118 a, 118 b over thetime of the injection. Further, since different solvents or mobilephases may have different compressibilities, the pressure may vary overlonger-time periods when mobile phase composition is varied, such as ingradient elution. Discontinuous or varying pressure may influence therate at which some components elute. Thus, it would be advantageous tohave a pump configured to provide a single shot stroke per sampleinjection to yield a more controlled sample flow, constant pressure overthe injection time, smaller dead volume, and more consistent pressureacross multiple samples and assays.

One such suitable pump 124 according to one embodiment of the presentinvention is briefly described below, is shown in to FIGS. 9A-9E, and isdescribed in greater detail in U.S. Provisional Application No.61/552,955, entitled “Syringe Pump,” Attorney Docket No. TFS-13BP, filedon even date herewith, and the disclosure of which is incorporatedherein by reference in its entirety.

Referring to FIGS. 9A and 9B, the pump 124 includes a piston pump 1250having a barrel 1252 and a piston 1254 in slidable relation with thebarrel 1252 and coupled to a housing 1256. The piston 1254 is driven bya motor 1258, such as a stepper motor, via a drive belt 1260 and a leadscrew 1262. More specifically, the motor 1258 drives a first pulley1264, which is mechanically-coupled to a second pulley 1266 by the drivebelt 1260. Rotation of the drive belt 1260 may be monitored, if desired,by a rotation sensor 1268. Suitable materials from which the piston 1254may be fabricated are, for example, various ceramics, including zirconiaand synthetic sapphire.

The lead screw 1262 is operably coupled to the second pulley 1266 suchthat rotation of the second pulley 1266 rotates the lead screw 1262. Tomaintain stability of the lead screw 1262 relative to the piston 1254,the lead screw 1262 may be rotatably coupled to a piston chamber 1270 ateach end 1272, 1274 of the lead screw 1262 and which is, in turn,coupled within the housing 1256. The lead screw 1262, between the pointsat which the lead screw 1262 is coupled to the piston chamber 1270,receives a threaded flange nut 1276 such that rotation of the lead screw1262 is configured to cause a linear movement of the threaded flange nut1276 along the lead screw 1262 and between the first and second ends1272, 1274.

An alignment plate 1278 is operably coupled to the threaded flange nut1276 for transferring the linear movement of the flange nut 1276 to thepiston 1254. The alignment plate 1278 may include three bores: a firstbore 1280 is operably coupled to the piston 1254 via a flexible joint1282, which is described in greater detail below, and each of second andthird through bores 1284, 1286 receives a respective guide pin 1288,1290. The guide pins 1288, 1290 are secured to the piston chamber 1270and provide structural stability and reciprocating-direction guidance(indicated by axis 1292) to the piston 1254.

The flexible joint 1282 coupling the piston 1254 to the alignment plate1278 allows for some movement of the piston 1254 relative to the barrel1252. In this way, the piston 1254 may self-correct its position and/orangular alignment with respect to the barrel 1252 during operation ofthe pump 124, reducing the amount of wear on the seal formed between thepiston 1254 and the barrel 1252, and providing for both a more efficientstroke and a more continuous output flow. In the illustrative embodimentof the flexible joint 1282, an adaptor end 1294 of the piston 1254 iscoupled to an insert 1296, which is operably secured within the firstbore 1280 of the alignment plate 1278, for example, by a threaded nut1298.

A semi-compliant ball, compressible, bearing 1300 is positioned betweenthe adaptor end 1294 and the insert 1296. In a rest state, the ballbearing 1300 is fully expanded and the adaptor end 1294 is biased awayfrom the insert 1296. However, atypical movement the piston 1254 duringoperation of the pump 124, for example, jamming the piston 1254 suchthat the piston 1254 is resisted from moving into the barrel 1252,creates a force on the piston 1254. The force compresses the ballbearing 1300 between the adaptor end 1294 and the insert 1296, whichreduces the stress placed onto the seal. A bumper 1301 may also beprovided for buffering the force created when the piston 1254 isexcessively drawn in the rearward direction.

By selecting the features of the lead screw 1262, the threaded nut 1298,and the motor 1258, the pump 124 may be configured to provide sufficientstroke volume such that a single stroke of the piston 1254 displaces asufficient volume of fluid to complete a single LC injection inaccordance with the selected assay or to elute an entire sample from theLC column 114, 116 in accordance with a chromatography system fluid flowrate and working flow pressure. The flow rate may need to be variablewithin a range from about 0 mL per minute to about 3 mL per minute. Theworking fluid pressure may vary between systems and experiments. Theworking fluid pressure may vary between systems and experiments. In somesituations, the maximum working fluid pressure may be about 100 bars. Inother systems, the maximum working fluid pressure may be about 200 bars,about 400 bars, about 600 bars, about 800, about 100 bars, or about 1500bars. By configuring the features, such as the torque, of the pump 124accordingly, the pump 124 may be designated to operate at a certainmaximum pressure. Generally, conventional pumps that are designed tooperate at higher fluid pressures may also operate at lower pressures.For example, by configuring the features of the instant pump 124accordingly—a force of approximately 900 pounds applied to a 7 mmdiameter piston 1254—a pressure of about 1000 bar (about 15000 poundsper square inch) would correlate to a volume displacement of about 3 mL.Generally, the piston diameter may range from about 4 mm to about 10 mm.

Other suitable configurations may include, for example, a motor 1258having a working load capacity of about 900 pounds and a thread pitch ofthe lead screw 1262 ranging from about 2 mm to about 20 mm, wherein thepitch is measured as an amount of linear travel per 360-degrees ofrotation of the lead screw 1262. Suitable stepper motors include, forexample, those that are commercially-available from ElectroCraft, inc.(Dover, N.H.) and their equivalents. Furthermore, air bubbles formedwithin one or more of the fluid lines may be passed during a singlestroke and eliminates the need for otherwise clearing the fluid lines.

In that regard, one method of using the pump 124 in accordance with oneembodiment of the present invention is described with reference to FIGS.9B and 9C. More specifically, in FIG. 9B, the piston 1254 is in a fullyretracted position and the barrel 1252 is filled with a volume of fluid,generally a solvent or a mobile phase. The motor 1258 is then operatedso as to cause the threaded flange nut 1276 and the alignment plate1278, and thus the piston 1254, to advance further into the barrel 1252,as shown in FIG. 9C. As a result, at least a portion of the volume ofthe fluid is ejected from outlet 1302 of the barrel 1252 and enters afluid conduit 1304 connecting the outlet 1302 with a first port 1306 aof a rotary valve 1308. The rotary valve 1308 may be a multi-portinjection valve that is known and used by those of ordinary skill in theart of liquid chromatography, and may include, for example, thecommercially-available RHEODYNE TITANHT, 6-position, 7-port rotary valve(Rheodyne, LLC, Rohnert Park, Calif.). Accordingly, the rotary valve1308 may include any number of auxiliary ports, though four of fiveauxiliary ports 1306 b, 1306 c, 1306 d, 1306 e are shown in the specificillustrative embodiment. A sixth port 1306 f is directed to an exit port1312, which is coupled to a multi-port valve 126 a, 126 b (FIG. 4B)(described in greater detail below) via a fluid conduit 1310.

An internal channel (not shown) within the rotary body 1314 isconfigured to creates fluid communication between the first port 1306 aand one of the auxiliary ports 1306 b, 1306 c, 1306 d, 1306 e or theexit port 1312 via the sixth port (not shown). In that regard, therotary body 1314, a motor 1316 (generally, a stepper motor) is operablycoupled to the rotary body 1314 and configured to rotate the rotary body1314 and to align the internal channel with a select one of the ports1306 b, 1306 c, 1306 d, 1306 e.

In the ejection mode, that is, when the piston 1254 advances to ejectthe fluid, the internal channel is rotated so as to fluidically couplethe first port 1306 a to the sixth port 1306 f. The fluid thus exits thepump 1308 and enters the multi-port valve 126 a, 126 b (FIG. 4B).

At the end of the stroke, as shown in FIG. 9C, the piston 1254 is thenretracted to the position shown in FIG. 9B. Prior to retraction, theinternal channel of the rotary body 1314 is rotated such that the firstport 1306 a is fluidically coupled to one of the auxiliary ports 1306 b,1306 c, 1306 d, 1306 e. Each of these auxiliary ports 1306 b, 1306 c,1306 d, 1306 e includes a fluid conduit 1318, which lead to a respectivesolvent or mobile phase container 122 (FIG. 3A) or possibly to a samplesource or sample loop. While not necessary, each mobile phase container122 (FIG. 3A) may contain a different mobile phase for conducting adifferent one of the assays. In any event, the internal channel isaligned to the select one of the auxiliary ports 1306 b, 1306 c, 1306 d,1306 e such that the barrel 1252 is refilled with the appropriate mobilephase or sample.

Once the internal channel is so configured, the barrel 1252 is filledwith the appropriate mobile phase by operating the motor 1258 so as toretract the piston 1254 and to the position shown in FIG. 9B. Retractionof the piston 1254 draws mobile phase liquid from the aligned auxiliaryport 1306 b, 1306 c, 1306 d, 1306 e, through the internal channel,through the outlet 1302, and into the barrel 1252. The rotary body 1314is then rotated and prepared for ejection of the mobile phase or sample.

It would be readily understood that when the mobile phase is change,that is from a first mobile phase for a first selected assay, to asecond mobile phase for a second selected assay, then multiple strokesof the piston 1254 may be necessary to sufficiently fill the barrel 1252with the appropriate mobile phase. In that regard, fluid ejected fromthe barrel 1252 during a flushing process may be directed to the wastecontainer 134 (FIG. 3C) for disposal.

Another embodiment of a pump 124′ suitable for use within the presentsystem 10 (FIG. 1) is shown and described with reference to FIGS. 9F-9G,and where similar numbers with primes refer to similar features.Specifically, in FIG. 9F, the lead screw 1262′ is positioned in-linewith the piston 1254′ and not vertically offset as was shown in FIG. 9B.Additionally, the motor 1258′ is operably coupled to the lead screw1262′ and positioned in-line with both the lead screw 1262′ and thepiston 1254′.

At least one guide pin (two guide pins 1288′, 1289′ are shown) flanksthe axis 1292′ defined by piston 1254′ and is coupled to the piston1254′ by an alignment plate 1278′. As was described in detail above, thealignment plate 1278′ with the guide pins 1288′, 1289′ are configured tomaintain a horizontal alignment of the piston 1254′ during a stroke.

In operation, the lead screw 1262′ extends through, and is threadablyconnected with a stationary plate 1320 proximate the first end 1272′ ofthe lead screw 1262′ while the piston 1254′ is structurally secured tothe second end 1274′ of the lead screw 1262′. Operation of the motor1258′ causes rotation of the lead screw 1262′ with respect to thestationary plate 1320. Because the stationary plate 1320 is securedwithin the housing 1256′, rotation of the lead screw 1262′ causes alinear movement of the same, which drives the piston 1254′ with respectto the barrel 1252′. The mobile phase may be loaded and ejected in thepump 124′ in a manner that is similar to the method described above withrespect to the pump 124 (FIG. 9A).

The volume of fluid displaced during a single stroke of the piston 1254is directed to one valve 126 a, 126 b for driving the sample injections.In that regard, and turning now to FIGS. 10A and 10B, the details of thevalves 126 a, 126 b are shown and described in greater detail. Thevalves 126 a, 126 b are injection valves having six ports (illustratedas “1” through “6”) each fluidically coupled to one injection port 104a, 104 b one or more of the mobile phase supplies 122 (FIG. 3A), a wastecontainer 134 (FIG. 3A), the columns 114, 116 (FIG. 7A), and a fluidloop 132 connecting Port-2 to Port-5. Each valve 126 a, 126 b furtherincludes a rotatable center 128 having three internal channels 130 forcoupling two adjacent ports, as shown. The valves 126 a, 126 b have twoconfigurations: “in line” and “fill in loop.” FIG. 10A illustrates the“in-line” position where the mobile phase from at least one of themobile phase supplies 122 (FIG. 3A) is directed via the pump 124 (FIG.9A) and the fluid line into Port-3. The rotatable center 128 is suchthat the mobile phase moves through one internal channel 130 to Port-2where the mobile phase fills, or pressurizes, the loop 132 and entersPort-5. The mobile phase then moves through another internal channel 130to Port-4 and is directed to the columns 114, 116 (FIG. 7A). Meanwhile,the appropriate injection port 104 a, 104 b is fluidically coupled toPort-1 and, via an internal channel 130, to the waste container 134(FIG. 3A) via Port-6.

Rotation of the rotatable center 128 places the valve 126 a into a “fillin loop” position, which is illustrated in FIG. 10B. As shown, themobile phase now moves through an internal channel 130 to Port-4 and outto the columns 114, 116 (FIG. 7A). The prepared sample, on the otherhand, enters the valve 126 a via Port-1 and is directed by an internalchannel 130 out to loop 132 so as to fill loop 132. The sample moves outof loop 132 at Port-5 and is directed, via another internal channel 130,to the Port-6 and then to waste container 134 (FIG. 3A). Once asufficient volume of the prepared sample has been introduced, forexample, three-times the volume of the loop 132, the rotatable center128 is again rotated to transition the valve 126 a, 126 b to the“in-line” position of FIG. 10A. Accordingly, the prepared sampleremaining within the injection port 104 a, 104 b is directed to thewaste container 134 (FIG. 3A) while the mobile phase enters the loop132, thereby displacing the prepared sample from the loop 132 and intothe columns 114, 116 (FIG. 7A). Continued flow of the mobile phaseflushes all of the prepared sample from the loop 132 and into thecolumns 114, 116 (FIG. 7A).

While not specifically shown, it will be appreciated that theappropriate injection port 104 a, 104 b may then receive a flush solvent(e.g., from a solvent container, not shown) to flush the injection port104 a, 104 b, the fluid line, the appropriate internal channels 130, andthe loop 132 in preparation for receiving the next prepared sample andreducing the likelihood of cross contamination.

It would be readily appreciated, and indeed is explained in greaterdetail below, that the rotation of the rotatable center 128 of thevalves 126 a, 126 b may be controlled by logic and timed in accordancewith a scheduler.

Turning again to FIGS. 3A, 3B, and 4B, the sample analysis station 24includes a separate injection port 156 that is associated with the valve133 and therefore by-passes the LC channels 118 a, 118 b. The injectionport 156 may be used for injecting a calibration standard or a controlstandard (illustrated throughout as “QC”) for performing a calibrationor control analysis respectively as appropriate. In some embodiments, acalibration solution supply 158 (FIG. 3B) may be provided near theinjection port 156 for ease of access when a calibration standard isnecessary. For clarity of discussion of the mass spectrometer 120hereafter, only the prepared sample injections will be discussed. Yet itwill be understood that a sample calibration standard or controlstandard injected via the injection port 156 would be analyzed in asimilar manner.

The injected, prepared sample moves through the columns 114, 116 in aknown manner such that at least one of the analytes of interest willelute off the columns 114, 116 at a retention time that differs from theretention time of other analytes of interest and/or the matrixcomponents, i.e., eluents. The eluents and analytes from both of thefirst and second LC channels 118 a, 118 b are directed into the valve133 where the eluents are directed into the waste container 134 whilethe analytes are directed to an ionization source 140 of the massspectrometer station 112. Alternative methods of sample introduction mayinclude, but are not limited to, on-line methods (such as, flowinjection analysis, direct infusion, and injection loops) and off-linemethods (such as, solid phase extraction, blood spot surface coatings,matrix-assisted laser desorption/ionization (“MALDI”) plate coatings, orcoatings on general surfaces), may also be used to introduce the sampleto the mass spectrometer 120.

As shown in FIG. 4B, an atmospheric pressure ionization (eitherelectrospray ionization (“ESI”) or atmospheric pressure chemicalionization (“APCI”)) device (referred to generally herein as “nebulizingionizer”) is used for ionizing the analytes received by the ionizationsource 140. In that regard, the nebulizing ionizer includes a capillary,probe, or needle (referred hereinafter as “needle” 142) having a solventconduit therein (not shown) and surrounded by a gas conduit therein (notshown). An outlet of the gas conduit is positioned about 0.1 mm to about0.2 mm proximally to an outlet of the solvent conduit. In ESI operationa voltage generator 144 is electrically coupled to the needle 142 and isoperable to create a high voltage difference between the needle 142 andthe counter-electrode that is either at the mass spectrometer 120.

In use, a solvent is supplied to the solvent conduit at a rate rangingfrom about 400 μL/min to about 2000 μL/min; however, one of ordinaryskill in the art will readily appreciate that the solvent supply varieswith the particular ionization source 140 selected. The particularsolvent used is dependent on the chemical nature of the analyte instudy, and the methodology for selection of an appropriate solvent iswell known to those of ordinary skill in the art. A gas, typically aninert gas, such as N₂, is supplied to the gas conduit at pressuresranging from about 0 bar to about 7 bar. The voltage generator 144 isactivated and provides a voltage potential, typically ranging from about−5 kV to about 5 kV, to the solvent within the needle 142.

The solvent traverses the solvent conduit to the solvent conduit outlet.There, the charged solvent is impacted by the surrounding high-pressuregas leaving the gas conduit outlet. This high-pressure gas, togetherwith formation of a Taylor cone (not shown) at the needle tip under theinfluence of the electric field, causes the flow of the charged solventto be nebulized into a spray of charged, nebulized solvent which maycontain one or more analytes of interest eluted from the columns 114,116 (FIG. 7A).

In APCI operation, the voltage generator 144 is electrically-coupled toa corona discharge electrode 145 positioned distal to the outlets,instead of to the needle 142. The high voltage applied to the coronadischarge electrode 145, if present, is operable to ignite a plasmawhich aids in the ionization of the nebulized solvent; however otherionization methods may be used and are generally known in the art. Theplasma causes the ionization of the solvent and analytes(s), and aportion of the charged solvent/analyte(s) will enter into the massspectrometer 120 as gas phase ions of the analytes (“gas phase ions”). Aion source that is switchable between ESI and APCI modes is described inco-pending U.S. Provisional Application No. 61/408,034, entitled“Combined Ion Source for Electrospray and Atmospheric Pressure ChemicalIonization,” naming inventors Hardman, Dunyach, Atherton, and Belford,filed on Oct. 29, 2010, and U.S. application Ser. No. 13/280,069, filedon even date here with, the disclosure of which are incorporated hereinby reference in their entireties.

It would be readily appreciated that other ionization techniques areknown and may be implemented as necessary or desired. For instance,ionization sources 140 suitable for ionization of liquid samples mayinclude, for example, heated electrospray ionization (“HESI”), nanosprayionization (“NSI”), thermospray, sonic spray, atmospheric pressurephotoionization (“APPI”), laser diode thermal desorption (“LDTD”),atmospheric sampling glow discharge ionization source (“ASGDI”),paperspray ionization techniques capable of extracting a specimen from adried bloodspot, and inductively-coupled plasma (“ICP”). Ionizationsources 140 that are suitable for ionization of gas samples may include,for example, chemical ionization (“CI”), electron impact (“EI”),resonance enhanced multi-photon ionization (“REMPI”), resonancemulti-photon detachment (“RMPD”), glow discharge, and spark ionization.Furthermore, the ionization sources 140 for gas samples may be adaptedfor use with liquid samples. Ionization sources 140 that are suitablefor desorbtion and ionization of a sample from a surface include, forexample, MALDI, surface-assisted laser desorption/ionization (“SALDI”),surface-enhanced laser desorption/ionization (“SELDI”), desorptionelectrospray ionization (“DESI”), direct analysis in real time (“DART”),discontinuous atmospheric pressure interface (“DAPI”), laser diodethermal desorption (“LDTD”), and field desorption. This listing ofpossible ionization sources 140 is not exhaustive and may include otherionization sources and/or permutations as would be readily understood bythose of ordinary skill in the art of mass spectroscopy and analyticalchemistry.

A skimmer 160, positioned distal to the corona discharge electrode 145,acts in conjunction with an auxiliary gas (not shown, but directedbetween the outlets and the skimmer 160) to contain and/or focus the gasphase ions into a vacuum chamber of the mass spectrometer 120. Theauxiliary gas may be supplied at rates that range generally from about 0L/min to about 15 L/min.

Referring still to FIG. 4B, the illustrative example of the massspectrometer 120 includes an interface 146 with the ionization source140, a mass filter 148, and an ion detector 150. The regions containingthe mass filter 148 and the ion detector 150 are maintained undervacuum. This interface 146 includes an orifice 152 of a skimmer cone 154that provides an opening into a higher vacuum chamber containing themass filter 148 while maintaining vacuum pressures.

In the illustrated example, the mass filter 148 is shown to be aconventional quadrupole; however, those skilled in the art willunderstand the determination by which the appropriate mass filtermodality for a given assay is selected. In fact, other mass spectrometerembodiments may include, for example, a single quadrupole modalities,time-of-flight (“TOF”) or exactive modalities, ion trap (“OT”)modalities, hybrid modalities, such as Q-TOF, TOF-TOF, Q-Exactive,LTQ-orbitrap, and LTQ-FT, or a mass spectrometer modified for protontransfer.

Single quadrupoles offer the benefits of small size, simple operation,and low cost; however, single quadrupoles lack analyte specificity andhigh back-ground noise levels, which lead to poor detection limits. TOFand Exactive modalities provide the benefits of greater selectivity ofthe gas phase ions for identification of analytes and may lead to thepossibilities of archiving data for later review and identification ofunknown analytes. However, as compared to quadrupole modalities, TOF andExactive modalities are larger, more expensive, and require highervacuums without gaining much decrease in the background noise levels.

Hybrid modalities, as compared with quadrupoles, TOF, and Exactivemodalities, provide greater specificity for parent/fragment gas phaseion pairs with confirmation by accurate mass measurements, and, as tothe TOF and Exactive modalities, the hybrid mass spectrometers may beoperated in a manner that is similar to the TOF and Exactive modalities.However, hybrid modalities are much larger, more temperature sensitive,require a higher vacuum, are more expensive, and require higher powerthan quadrupole, TOF, and Exactive modalities.

Ion trap modalities allow the consideration and summation of multiplefragments for analyte quantification, which is particularly advantageousfor vitamin D and testosterone-type assays, and increased massspecificity. However, these modalities tend to much more expensive,require higher vacuum and power, and are larger than the other availablemodalities.

Still other modalities are available, and may include, for example, ioncyclotron resonance (“ICR”) or magnetic sector-based analyzers. Whilethese modalities offer high resolution and specificity, each is alsovery expensive. Furthermore, other detectors/analyzers may be used with,or in place of the mass spectrometer. For example, these detectors mayinclude, for example, electrochemical devices, nuclear magneticresonance (“NMR”), absorbance, fluorescence, refractive index, pH,and/or conductivity.

In the illustrated quadrupole mass filter modality, an ion current isdirected through four parallel electrodes comprising the mass filter148, wherein the four parallel electrodes are comprised of two pairs ofelectrodes. A radiofrequency field and a DC voltage potential areapplied to each of opposing pairs of electrodes by appropriate powersupplies such that the two pairs are 180 degrees out of phase in RFvoltage and differ in polarity of DC voltage potentials. Only those ionswithin the ion current having a first mass-to-charge ratio, m1/z1, willcontinue through the parallel electrodes to the ion detector 150. Saidanother way, the m1/z1 ions will be equally attracted to and deflectedby the two pairs of electrodes while the mean free path induced by theradiofrequency field onto the m1/z1 ions does not exceed the distancebetween the electrodes. Thus, the m1/z1 ion will balance theradiofrequency and DC voltage forces from the parallel electrodes andtraverse the parallel electrodes to impact the ion detector 150.

The m1/z1 ions that reach the ion detector 150, which may be in oneembodiment, an electron multiplier (“EM”) having a plurality of dynodes.In some embodiments, the EM may be replaced by a photomultiplier tube(“PMT”) so as to detect photons produced by secondary electronsimpinging on an optional phosphorescent screen (not shown). Each m1/z1ion entering the EM strikes a first dynode and produces secondaryelectrons, which strike a third dynode to produce more secondaryelectrons, and so forth. The cascade of secondary electrons is finallycaptured by the multiplier collector, typically an anode, and measuredas a current (I) induced by a total number (n) of secondary electronsover a measured detection time (t) and in accordance with n/t=I/e,wherein e is the elemental charge. In other embodiments, the iondetector 150 may include a phosphorescent screen where the m1/z1 ionscollide with an electrode plate to produce secondary electrons that aredirected to the phosphorescent screen. The secondary electrons incidenton the phosphorescent screen produce photons that are detected by a PMT.

Analysis by way of a quadrupole mass filter 148 continues with alteringthe operational conditions of the mass filter 148 such that ions havinga second mass-to-charge ratio, m2/z2, will traverse the mass filter 148and impact the ion detector 150 in the manner similar to what was justdescribed for m1/z1 ions. A spectrum may then be generated relating theion flux, total ion current (“TIC”), or normalized relative abundanceswith respect to m/z of the ions detected.

In some embodiments, the resolution of the MS technique may be enhancedby employing “tandem mass spectrometry” or “MS/MS” for example via useof a triple quadrupole mass spectrometer. In those embodiments using atriple quadrupole, such as, for example, the mass-spectrometer describedin greater detail in U.S. Pat. No. 6,987,261, entitled “Controlling IonPopulations in a Mass Analyzer,” assigned to Thermo Finnigan, LLC,naming inventors Horning, Malek, Syka, and Wieghaus, and the disclosureof which is incorporated herein by reference in its entirety, threequadrupole stations are aligned in series. According to one embodimentin accordance with this technique, a first quadrupole station isoperated to generate a first ion (also referred to as a parent ion or aprecursor ion by those skilled in the art) having a particular m/z. Inthis way, the first quadrupole station may be used as a high pass filterto eliminate interfering substances, which is particularly useful incomplex samples, such as samples prepared from biological or patientspecimens. Suitable first quadrupole stations may include an ion trap orother mass filter modalities as described previously.

The first ion may then pass to a second quadrupole station, such as afragmentation chamber, in which the first ions are fragmented into oneor more second ions (also known as product ions or fragment ions).Accordingly, the second quadrupole station may be a collision cellwherein the incoming first ions collide with, and are fragmented by,neutral gas molecules (e.g., Ar). This process is called CollisionActivated Dissociation (“CAD”). In other embodiments where fragmentationis not necessary, the second quadrupole station may be a second ion trapor mass filter.

The second ions then enter a third quadrupole station, often a massfilter or ion trap, where the second ions corresponding to the analyteof interest are separated from ions of no immediate diagnostic interest.

Each of the quadrupole stations may be operated in triple quadrupole inone of several modes, including: a scan mode (scan or transmission ofselect m/z ions), pass mode (wide range of m/z ions permitted to pass),a fragmenting mode (collision of ions with an inert gas causingfragmentation), or a set mode (ions of a single m/z ions valuetransmitted). Selection of any one of these modes permits variousoperational parameters for analysis of the prepared sample in accordancewith the predetermined assay.

Turning now to the hardware and software environments of the system 10,FIG. 11 is a diagrammatic illustration of a hardware and softwareenvironment for the sample preparation controller 22 consistent withembodiments of the present invention. In specific embodiments, thesample preparation controller 22 is a computer, computing system,computing device, server, disk array, or programmable device such as amulti-user computer, a single-user computer, a handheld computingdevice, a networked device (including a computer in a clusterconfiguration), a mobile telecommunications device, a video game console(or other gaming system), etc. As such, the sample preparationcontroller 22 is referred to hereinafter as “prep controller” 22.

The prep controller 22 includes at least one central processing unit(“CPU”) 300 coupled to a memory 302. Each CPU 300 is typicallyimplemented in hardware using circuit logic disposed on one or morephysical integrated circuit devices or chips. Each CPU 300 may be one ormore microprocessors, micro-controllers, field programmable gate arrays,or ASICs, while memory 302 may include random access memory (“RAM”),dynamic random access memory (“DRAM”), static random access memory(“SRAM”), flash memory, and/or another digital storage medium, and alsotypically implemented using circuit logic disposed on one or morephysical integrated circuit devices, or chips. As such, the memory 302may be considered to include memory storage physically located elsewherein the prep controller 22, e.g., any cache memory in the at least oneCPU 300, as well as any storage capacity used as a virtual memory, e.g.,as stored on a mass storage device 304 or as stored on the LIS 28 (FIG.2) coupled to prep controller 22 through at least one network interface306 (illustrated as, and hereinafter, “network I/F” 306) by way of theat least one network 30. It will be appreciated that the at least onenetwork 30 may include at least one private communications network(e.g., such as an intranet) and/or at least one public communicationsnetwork (e.g., such as the Internet).

The prep controller 22 is coupled to at least one peripheral devicethrough an input/output device interface 308 (illustrated as, andhereinafter, “I/O I/F” 308). In particular, the prep controller 22receives data from a user through at least one user input device 310(including, for example, a keyboard, mouse, a microphone, and/or otheruser interface) and/or outputs data to the user through at least oneoutput device 312 (including, for example, a display, speakers, aprinter, and/or another output device). Moreover, in some embodiments,the I/O I/F 308 communicates with a device that is operative as a userinput device 310 and output device 312 in combination, such as a touchscreen display 313 (FIG. 1).

In addition to the network I/F 306 and the I/O I/F 308, the prepcontroller 22 may include a system interface 314 (illustrated as “systemI/F” 314). The system I/F 314 is connected to at least one communicationlink to the sample analysis controller 26 as well as at least onecommunication link to the sample preparation station 20. In particular,the system I/F 314 provides both a high level data interface link, asindicated at arrow 315 a (e.g., a TCP/IP link) or other common link, anda low level data interface link, as indicated at arrow 315 b (e.g., aCAN BUS, a uni-directional contact closure communication link, or abi-directional I/O level communication link) to the sample analysiscontroller 26. The system I/F 314 also provides a low level datainterface link (e.g., a contact closure, CAN BUS, or other I/O levellink, as indicated at arrow 315 c) to the sample preparation station 20for control by the prep controller 22. As such, the prep controller 22is configured to provide commands and other instructional data to thesample analysis controller 26 and receive results information via theTCP/IP link 315 a. Moreover, the prep controller 22 is configured toprovide low level data commands to control the sample analysis station24 (FIG. 2) and receive status information to track the operation of thesample analysis station 24 (FIG. 2) or control the sample analysisstation 24 (FIG. 2) via the I/O level link 315 b.

The prep controller 22 is typically under the control of an operatingsystem 316 (illustrated as “OS” 316) resident in the memory 302 andexecutes or otherwise relies upon various computer softwareapplications, sequences of operations, components, programs, files,objects, modules, etc., consistent with embodiments of the invention. Inspecific embodiments, the prep controller 22 executes or otherwiserelies on at least one preparation control application 318 (referred tohereinafter as “control application” 318) to manage the operation of thesample preparation station 20 and monitor the sample analysis station 24(FIG. 2), as well as process the results provided by the sample analysisstation 24 (FIG. 2).

FIG. 12 is a diagrammatic illustration of a plurality of applications,sequences of operations, components, programs, files, objects, modules,etc. (each of which is referred to hereinafter as a “module” forsimplicity), that may be included in the control application 318 of FIG.11. Specifically, the control application 318 may include one or more ofeach of the following modules: a LIS interface module 320 (illustratedas “LIS I/F” 320); a user interface module 322 (illustrated as “userI/F” 322); a sample analysis station client module 324; an informationacquisition device module 325; a mixer module 326; a centrifuge module328; an optional vessel rack loader module 330 (such as for the system10′ of FIG. 3B); a waste control module 332; a tray loader module 334; awash solution control module 336; a robotics module 338; a cover/lockcontrol module 340; a pipetter module 342; an injector module 344; asyringe pump module 345; a turntable module 346; a mobile phase controlmodule 348; and a sample preparation module 350. The sample preparationmodule 350, in turn, may include a scheduler module 352, a resultsprocessing module 354, and a reporting module 356.

With respect to the individual modules, the LIS I/F module 320 isconfigured to control communications between the prep application 318and the LIS 28 (FIG. 2), while the user interface module 322 isconfigured to provide human perceptible outputs to a user (not shown)(e.g., such as audibly and/or visually perceptible sounds and/or imageswith the output device 312) (FIG. 11). The sample analysis stationclient 324, on the other hand, is configured to send data (such ascommands and other instructional data) and receive data (such asresults) respectively to and from the sample analysis controller 26(FIG. 2) via the high level data link 315 a. The information acquisitiondevice module 325 is configured to control the operation of theinformation acquisition device 54 (FIG. 3A). The mixer modules 326 andthe centrifuge module 328 are configured to control at least a portionof the secondary processing station 80 (FIG. 3A), including one or morerespective mixing stations 82 (FIG. 3A) and/or centrifuges 88 (FIG. 3A).The vessel loader module 330, if present, is configured to control thetransport assembly 60 (FIG. 3A) to load vessels 58 (FIG. 3B) from thestorage station 59 (FIG. 3D) or another portion of the secondaryprocessing station 80 (FIG. 3A).

The sample preparation station 20 (FIG. 2) often produces waste as aresult of the preparation of the samples. Such waste may include aportion of a sample that was not analyzed by the sample analysis station24 (FIG. 2), waste produced by cleaning the sample pipette assembly 62(FIG. 4A), cleaning the injector pipette assembly 96 (FIG. 3A), clearingthe sample preparation station 20 (FIG. 2) of the vessels 58 (FIG. 3A)that are no longer necessary, and/or clearing the sample preparationstation 20 (FIG. 2) of samples that are no longer necessary. As such,the control application 318 includes the waste control module 332 whichoperates to clear the sample preparation station 20 (FIG. 2) of thatwaste. The tray loader module 334 is configured to control the transportassembly 60 (FIG. 3A) to load at least one vessel 58 of the vessel rack84 (FIG. 3B), if used, to the injector station 92 (FIG. 3A), while thewash solution control module 336 monitors the amount of wash solutionfrom the flush solvent container (not shown) and controls the dispensingof that wash solution. The robotics module 338, on the other hand,controls the movement of the transport assembly 60 (FIG. 3A) generally(including any grippers used to transport vessels 58 (FIG. 3A) and/orvessel racks 84 (FIG. 3B), if used, within the system 10 (FIG. 1)),while the cover/lock control module 340 ensures that one or more covers357 a, 357 b (FIG. 1) of the system 10 (FIG. 1) are closed and lockedwhile the system 10 (FIG. 1) operates to prepare the samples. Theturntable module 346 controls the sampling station 56 (FIG. 3A) and therotatable tables 44, 102 (FIG. 3A) of the system 10 (FIG. 1), while themobile phase control module 348 is configured to monitor the level ofmobile phases within the mobile phase supplies 122 (FIG. 3A) todetermine if any volume thereof is low. In one embodiment, the system 10(FIG. 1) may include sensors (not shown) that are configured to indicatethe level of the mobile phases and communicate that information via thelow-level data link 315 b received by the injector module 344, while inan alternative embodiment the system 10 (FIG. 1) is configured todetermine how much of any particular mobile phase remains by calculatingthe amount of mobile phase used during testing.

With respect to the remaining modules, the pipetter module 342 controlsthe operation of the sample pipette assembly 62 (FIG. 4A), while theinjector module 344 controls the injector pipette assembly 96 (FIG. 4A)for injecting the prepared sample into the appropriate injector port 104a, 104 b (FIG. 3A). Moreover, the injector module 344 is configured toreceive status and/or other low-level data from the sample analysisstation 24 (FIG. 2), and provide commands and/or other low-level data tothe sample analysis station 24 (FIG. 2) though the system I/F 314 (FIG.11). For example, the injector module 344 may be configured to receivestatus information about individual components of the sample analysisstation 24 (FIG. 2), including whether a particular injector port 104 a,104 b (FIG. 3A), the valves 126 a, 126 b (FIG. 3A), and/or the massspectrometer 120 (FIG. 3A) is available or ready. The controlapplication 318 may in turn utilize that data to control the operationof the sample analysis station 24 (FIG. 2) via commands sent through thesample analysis station client module 324. A syringe pump module 345 isconfigured to receive instructions for, operate, and/or report thestatus of the pump 124 (FIG. 9A). For example, the syringe pump module345 may control a stroke volume, a stroke rate, report whether the pump124 (FIG. 9A) is available, report whether the pump 124 (FIG. 9A) isoperating properly, etc. Although not illustrated, one or more ofmodules 326-248 may be configured to communicate with their respectivecomponents through the low level data interface link 315 c (FIG. 11).

The sample preparation module 350 is configured to monitor and supervisethe operation of modules 320-348. In addition, the sample preparationmodule 350 is configured to schedule samples for preparation andanalysis by the sample preparation station 20 (FIG. 2) and the sampleanalysis station 24 (FIG. 2), respectively, with the scheduler module352. The scheduler module 352 is configured to determine an order inwhich to process specimens. For example, each specimen 23 (FIG. 2)received by the system 10 (FIG. 1) is associated with data used todetermine a target time to result and/or target time to prepare thatspecimen 23 (FIG. 2). The scheduler module 352, in turn, determines wheneach specimen 23 (FIG. 2) should be prepared and/or analyzed based uponthe specimen type, whether the specimen 23 (FIG. 2) is a priorityspecimen, the type of assay to perform, and the time for completionassociated with that specimen 23 (FIG. 2), as well as corresponding dataassociated with other samples and/or specimens 23 (FIG. 2) in the system10 (FIG. 1). Thus, a second specimen with a short time for completionmay be processed prior to a first specimen with a longer time forcompletion and received prior to that second specimen. The schedulermodule 352 thus provides the ability to dynamically adjust the order inwhich samples are prepared and analyzed by the system 10 (FIG. 1).

The sample preparation module 350 may further include a resultsprocessing module 354. The results processing module 354 is configuredto analyze results from the sample analysis station 24 (FIG. 2) anddetermine if those results are consistent with calibration and/orcontrol data. The results processing module 354 may also be configuredto format results for storage by the LIS 28 (FIG. 2). The reportingmodule 356, in turn, is configured to provide reports about operation ofthe system 10 (FIG. 1), such as event reports and alarm reports.

FIG. 13 is a diagrammatic illustration of a plurality of data structuresthat may be included in the mass storage device 304 of the prepcontroller 22 (FIG. 2). Specifically, FIG. 13 illustrates that the massstorage device 304 may include an assay data structure 360, ascheduler/status data structure 362, a calibration/control parametersdata structure 364 (illustrated as “CALIBRATION/QC PARAMETERS” 364), aresults data structure 366, a sample association data structure 368, anda log data structure 370. The assay data structure 360 may be a databaseconfigured to store information about each type of assay, and inparticular reagents or solvents that may be used to prepare a specimen23 (FIG. 2) for analysis, operations to prepare the specimen inaccordance with a particular assay, and/or information required by thesample analysis station 24 (FIG. 2) to perform the analysis. Moreover,the assay data structure 360 may store data about the types of assaysthat the system 10 (FIG. 1) may run.

Specifically, the system 10 (FIG. 1) may be configured to monitorwhether there is enough reagent for the particular type of assay andprevent that type of assay from being run when there is not enoughreagent. Moreover, the system 10 (FIG. 1) may be configured to determinewhich types of assays are associated with calibration data such thatdata associated with that type of assay may be determined. As such, theassay data structure 360 includes a cleared assay list 361 a indicatingtypes of assays for which the system 10 (FIG. 1) has sufficient reagentsand that are associated with a valid calibration, as well as a pendingassay list 361 b indicating types of assays for which the system 10(FIG. 1) has sufficient reagents but is associated with a calibrationthat is currently pending.

The system 10 (FIG. 1) will perform an assay in the cleared assay list361 a but refrain from performing an assay that is in the pending assaylist 361 b until a calibration curve for that assay has been generated.The assay data structure 360 also includes an invalid assay list 361 cindicating types of assays for which the system 10 (FIG. 1) does nothave sufficient reagents, or types of assays that are not associatedwith a calibration or that are associated with an invalid calibration.The system 10 (FIG. 1) will not prepare a sample in accordance with anassay in the invalid assay list 361 c until such time as that assay ismoved to the cleared assay list 361 a.

With respect to the remaining data structures, the scheduler/status datastructure 362 may store data used by the prep controller 22 (FIG. 2) todetermine the order in which to prepare specimens, data used by the prepcontroller 22 (FIG. 2) to determine the order in which to perform samplepreparations, and/or data indicating the status of each specimen 23(FIG. 2), sample, or prepared sample in the system 10 (FIG. 1), and/orthe testing to be performed by the system 10 (FIG. 1) for a particularsample. That data may be provided to the user for the user to view dataabout the specimens 23 (FIG. 2), prepared samples, and/or tests to beperformed by the system 10 (FIG. 2). The calibration/control parametersdata structure 364 stores data about calibrations and/or control resultsof the sample analysis station 24 (FIG. 2), while the results datastructure 366 may store the results of tests performed by the sampleanalysis station 24 (FIG. 2). The sample association data structure 368is configured to associate a specimen 23 (FIG. 2) with a particularresult or set of results. The log data structure 370 may store a log ofoperational data about the system 10 (FIG. 1), including events and/oralarms detected thereby.

FIG. 14 is a diagrammatic illustration of a hardware and softwareenvironment for the sample analysis controller 26 consistent withembodiments of the present invention. In specific embodiments, andsimilarly to the prep controller 22 (FIG. 2), the sample analysiscontroller 26 is a computer, computing system, computing device, server,disk array, or programmable device such as a multi-user computer, asingle-user computer, a handheld computing device, a networked device(including a computer in a cluster configuration), a mobiletelecommunications device, a video game console (or other gamingsystem), etc.

The sample analysis controller 26 includes at least one CPU 400 coupledto a memory 402. Each CPU 400 is typically implemented in hardware usingcircuit logic disposed on one or more physical integrated circuitdevices or chips. Each CPU 400 may be one or more microprocessors,micro-controllers, field programmable gate arrays, or ASICs, whilememory 402 may include RAM, DRAM, SRAM, flash memory, and/or anotherdigital storage medium, and also typically implemented using circuitlogic disposed on one or more physical integrated circuit devices, orchips. As such, memory 402 may be considered to include memory storagephysically located elsewhere in the sample analysis controller 26, e.g.,any cache memory in the at least one CPU 400, as well as any storagecapacity used as a virtual memory, e.g., as stored on a mass storagedevice 404, which may contain a log data structure 405 a to store dataassociated with the sample analysis controller 26 and/or sample analysisstation 24 (FIG. 2), and a configuration data structure 405 b which maystore information used to generate a methodology of how the sampleanalysis station 24 (FIG. 2) will prepare the sample. In one embodiment,the configuration data structure 405 b stores configuration data in theXML format. The configuration data structure 405 b may also indicateeach of the assays that are performable by the sample analysis station24 (FIG. 2). As such, the configuration data structure 405 b may beaccessed by the prep controller 22 or the sample analysis controller 26in response to a query from the prep controller 22 as to which assaysmay potentially be performed. A registered virtual interfaceconfiguration data structure 405 c (illustrated as “registered VIconfiguration(s)” 405 c) stores information about the configuration ofvirtual interfaces (FIG. 15) utilized by the sample analysis controller26.

The sample analysis controller 26 is coupled to the prep controller 22through at least one system interface 406 (illustrated as “system I/F”406). As such, the system I/F 406 may include appropriate circuitry tocommunicate across the high level data link 315 a and the low level datalink 315 b. The sample analysis controller 26 is coupled to at least oneperipheral device through an input/output device interface 408(illustrated as, and hereinafter, “I/O I/F” 408). In particular, thesample analysis controller 26 receives data from a user through at leastone user input device 410 (including, for example, a keyboard, mouse, amicrophone, and/or other user interface) and/or outputs data to the userthrough at least one output device 412 (including, for example, adisplay, speakers, a printer, and/or another output device). Moreover,in some embodiments, the I/O I/F 408 communicates with a device that isoperative as a user input device 410 and output device 412 incombination, such as the touch screen display 313 (FIG. 1). In specificembodiments, the sample analysis controller 26 is generally configuredto provide data to the prep controller 22 and not otherwise beaccessible through the user input device 410 or provide informationthrough the output device 412. However, in certain circumstances, suchas during debugging, maintenance, and/or administrative functions, itmay be desirable for users to be able to access the sample analysiscontroller 26 through the user input device 410 and for the sampleanalysis controller 26 to provide human perceptible output through theoutput device 412.

The sample analysis controller 26 is typically under the control of anOS 416 resident in the memory 402 and executes or otherwise relies uponvarious computer software applications, sequences of operations,components, programs, files, objects, modules, etc., consistent withembodiments of the invention. In specific embodiments, the sampleanalysis controller 26 executes or otherwise relies on at least onesample analysis application 418 (referred to hereinafter as “sampleanalysis application” 418) to receive commands from the prep controller22, operate the sample analysis station 24 (FIG. 2), and provide resultsback to the prep controller 22.

FIG. 15 is a diagrammatic illustration of a plurality of applications,sequences of operations, components, programs, files, objects, modules,etc. (each of which is referred to hereinafter as a “module” forsimplicity), that may be included in the sample analysis application 418of FIG. 14. The sample analysis application 418 includes a sampleanalysis station service module 420 that is configured to communicatevia the high level data link 315 a with the sample analysis stationclient module 324 (FIG. 12) of the control application 318 (FIG. 12).The sample analysis station service module 420 functions as the primarypoint of communication between the control application 318 (FIG. 12) andthe sample analysis application 418. As such, the sample analysisstation service module 420 may receive instructional data, including anindication of the assay type to perform, what analytes to detect withthe assay type, the amount of prepared sample necessary in accordancewith the assay, and a globally unique identification designator (“GUID”)of the prepared sample. Results for an analyzed sample will include theGUID of the specimen 23 (FIG. 2) for the system 10 (FIG. 1) to associatespecimens 23 (FIG. 2) and their results. The sample analysis stationservice module 420 is configured to analyze that instructional data anduse that data to look up, with a data manager 422, how to operate thesample analysis station 24 (FIG. 2) to prepare the same in accordancewith the assay. The sample analysis station service module 420, in turn,is configured to provide the results back to the sample analysis stationclient module 324 (FIG. 12).

The sample analysis application 418 also includes an acquisition service424 that operates to multiplex the operation of the sample analysisstation 24 (FIG. 2) and pass commands to the components of the sampleanalysis station 24 (FIG. 2). In particular, the acquisition service 424may be configured to operate in a modular manner with respect to aplurality of different sample analysis stations 24 (FIG. 2), and as suchcommunicate with components of the sample analysis station 24 (FIG. 2)through various virtual interfaces, each virtual interface beingspecific to one or more of those components. For example, and asdescribed above, the sample analysis station 24 (FIG. 2) may include themass spectrometer 120 that in turn includes pumps 124, and valves 126 a,126 b. As such, the acquisition service 424 communicates with amultiplexing virtual interface 426 (illustrated as, and referred tohereinafter, as a “MUX VI” 426) that translates modular data from theacquisition service 424 into data that is appropriate for the injectorpipette assembly 96, the pumps 124, and the valves 126 a, 126 b. Theacquisition service 424 similarly communicates with a mass spectrometervirtual interface 428 (illustrated as, and referred to hereinafter, as“MS VI” 428) that translates modular data from the acquisition service424 into data that is appropriate to operate the mass spectrometer 120.The MUX VI 426 and MS VI 428 also operate to translate data from therespective injector pipette assembly 96, the pumps 124, the valves 126a, 126 b, or the mass spectrometer 120 to data that may be understood bythe acquisition service 424, if necessary.

In particular, the MUX VI 426 translates modular commands to operate theinjector pipette assembly 96, the pumps 124, or the valves 126 a, 126 bfrom the acquisition service 424 into commands that are appropriate forthe firmware 430 of the injector pipette assembly 96 of the samplepreparation station 20 (which may be physically located at the sampleanalysis station 24, both shown in FIG. 2) as well as the firmware 430of the pumps 124 and the valves 126 a, 126 b of the sample analysisstation 24 (FIG. 2). Correspondingly, the MUX VI 426 translates dataspecific to the injector pipette assembly 96 and the pumps 124 and/orthe valves 126 a, 126 b (e.g., status data) into data that may beunderstood by the acquisition service 424, if necessary. Similarly tothe MUX VI 426, the MS VI 428 translates modular commands to operate themass spectrometer 120 from the acquisition service 424 into commandsthat are appropriate for the firmware 432 of the components of the massspectrometer 120, and also translates data from the mass spectrometer120 (e.g., result data) into data that may be understood by theacquisition service 424, if necessary.

As illustrated in FIG. 15, the MUX VI 426 is also configured to provideinformation to the injector module 344 (FIG. 12) of the controlapplication 318 (FIG. 12) via the low level link 315 b through thesystem interface 406 (FIG. 14). This low level link 315 b may carrystatus data from the components of the sample analysis station 24 (FIG.2), such as the status of the injector pipette assembly 96 or acomponent thereof, as well as the status of the one or more valves 126a, 126 b and the status of an analysis of a prepared sample. Thus, thecontrol application 318 (FIG. 12) may know the status of the sampleanalysis station 24 (FIG. 2) and may control at least some of theoperations thereof.

The sample preparation controller 22 (FIG. 2) is configured to provide auser interface of the system 10 (FIG. 1) to a user, such as the touchscreen display 313 (FIG. 1). The user interface may allow the user toinput data, such as data associated with particular specimens, as wellas view data about the system 10 (FIG. 1), including status informationand results of an analysis. In some embodiments, the system 10 (FIG. 1)does not require user input, but instead may automatically determinewhich assay to run by querying the LIS 28 (FIG. 2). Accordingly, theuser interface may be used for displaying system status and systemdiagnostics to a passive user.

FIGS. 16A-16G illustrate screenshots that may be provided by the samplepreparation controller 22 (FIG. 2) consistent with embodiments of thepresent invention. In particular, FIG. 16A illustrates a start screen500 that may be presented to a user, for example a laboratorytechnician, to enter information about a specimen. The start screen 500includes a test sample commands section 502 for the user to interactwith the system 10 (FIG. 1); a submitted samples section 504 fordisplaying a list that includes the ID, name, status, assay type, andschedule for specimens 23 (FIG. 2) loaded into the system 10 (FIG. 1); adevice status section 506 that provides information regarding the samplepreparation station 20 (FIG. 2) and the sample analysis station 24 (FIG.2), such as errors, statuses, and identification information thereof;and an acquisition commands section 508 for initiating, starting, orpausing the system 10 (FIG. 1). The user may also clear a log byselecting a clear log button 510.

In the test sample commands section 502, the user may select a submittest sample user interface component 523 to enter information regardinga particular specimen 23 (FIG. 2) and to start an analysis thereof. Inresponse to selection of the submit test sample user interface component523, the sample preparation controller 22 (FIG. 2) provides a setupsingle test sample component 512, illustrated in FIG. 16B. The setupsingle test sample component 512 allows the user to input particularinformation through user interfaces (e.g., each user interface being atext box, a selection box, a check box, or some other appropriate humanperceptible user interface), such as a sample name in a sample name userinterface 514, the type of assay to perform in an assay type userinterface 516, the time for the result of a sample analysis to beprovided in a time to result user interface 517, whether the specimen isa priority specimen through a priority user interface 518, a dilutionfactor in a dilution factor user interface 519, a volume of the samplein a volume user interface 520 (entered in mL), and an indication of theperson who submitted the specimen 23 (FIG. 2) in a submitter userinterface 522 (e.g., the name of the laboratory technician). The usermay then select an “OK” or “Cancel” button as appropriate and return tothe start screen 500.

If more than one test is ordered by a prescribing physician for aparticular specimen 23 (FIG. 2), the user may select a submit singletest user interface 524 to enter information for an additional test in amanner similar to that described in connection with FIG. 16B. The usermay also remove a particular job (i.e., test or sample) by selecting theappropriate specimen 23 (FIG. 2) or test within the submitted samplessection 504 and then selecting a remove selected job user interface 526.Selecting an update statuses user interface 528 that allows the user toretrieve current information on the status of each test of the specimen23 (FIG. 2) that is presently under analysis.

The user may also select a desired appropriate specimen 23 (FIG. 2) ortest within the submitted samples section 504 and then select a viewtests section 530 to open a test for sample window 532, illustrated inFIG. 16C, to display the status and results for a selected specimen 23(FIG. 2) or test. As shown, the test information component 534 providesvarious information about the prepared sample under analysis in a listand includes an indication of an assay ID used for the prepared sample,barcode for the prepared sample, assay selected for the prepared sample,the LC channel 118 a, 118 b (FIG. 4B) scheduled to receive that preparedsample, and the status of that prepared sample. Information regardingresult data from the analysis of that prepared sample may also bedisplayed, such as a peak area (i.e., “AA”) and an internal standard(“ISTD”) peak area. As illustrated in FIG. 16C, results for ahydrocortisone assay of vessel number fifty-three are being acquired.The user may select an update status user interface 536, get result userinterface 538, view result user interface 540, or close window userinterface 542, as appropriate. Selection of the close window userinterface 542 returns the user to the start screen 500 (FIG. 16A).

Returning again to FIG. 16A, the user may also select a “Who'sAcquiring” user interface 544 to have the system 10 (FIG. 1A) identifythe specimen 23 (FIG. 2) and/or assay for the specimen 23 (FIG. 2) fromthe submitted samples section 504 that is being acquired.

Once the analysis of the prepared sample has completed such that thedata related thereto has been gathered (e.g., the acquisition is“complete”), the status of the particular sample associated with thatdata indicated in the submitted samples section 504 is updated to“Complete.” The user may then select the particular sample and select aview tests user interface 530 to review the results.

FIG. 16D illustrates a test for sample window 532 that is provided oncean analysis has completed. Accordingly, the status under the informationblock has been updated to “Completed” and the calculated values for thehydrocortisone assay are provided, as appropriate, under peak area andISTD peak area. A chromatogram 546 is also displayed, specifically shownherein as illustrating the hydrocortisone results.

FIGS. 16E-16G illustrate a plurality of additional screens that may beprovided by the system 10 (FIG. 1) consistent with embodiments of thepresent invention. Specifically, one or more of the screens illustratedin FIGS. 16E-16G may be provided in addition to, or instead of, one ormore of the screens illustrated in FIGS. 16A-16D. In particular, FIG.16E illustrates a specimen information screen 550 that may be providedby the system 10 (FIG. 1) consistent with embodiments of the presentinvention. The specimen information screen 550 provides a specimensection 552 that indicates information associated with a specimen 23(FIG. 2) in the system 10 (FIG. 1), and in particular lists its locationwithin the system 10 (FIG. 1) and/or in a vessel 58 (FIG. 2) and/or aparticular vessel rack 84 (FIG. 3B), if used. The specimen section 552also provides a list of each specimen 23 (FIG. 2) in the system 10 (FIG.1). As such, a particular specimen 23 (FIG. 2) in the list can beselected and the information associated therewith viewed in a specimendata section 554. The specimen data section 554 indicates informationassociated with a selected specimen 23 (FIG. 2) as well as allows theuser to input data about that particular specimen 23 (FIG. 2). Inparticular, the user may specify the type of specimen in a specimen typecontrol 555, information associated with the specimen 23 (FIG. 2) (e.g.,such as a Time to Return results or a Time to Prepare the specimen 23)in a information control 556, as well as view the tests for the specimen23 (FIG. 2) and any results in a specimen test status control 557.Moreover, the user may designate a selected specimen as a priorityspecimen by selecting a “Priority” button 558.

As illustrated in FIG. 16E, the specimen information screen 550 may alsoincludes a position indicator 560 and, optionally, a rack indicator 559indicating the position and rack, respectively, of the specimen 23 (FIG.2) in the vessel 58 (FIG. 3A) or the vessel rack 84 (FIG. 3B). Thespecimen information screen 550 further includes a status indicator 561to indicate the status of the specimen 23 (FIG. 2) or prepared samplethereof. In one embodiment, the specimen information screen 550 furtherincludes a specimen interaction section 562 that may include a pluralityof buttons for the user to specify data associated with the specimen 23(FIG. 2), including the particular type of tests to perform on thespecimen 23 (FIG. 2).

FIG. 16F illustrates a specimen location screen 570 that graphicallyillustrates the locations of specimens 23 (FIG. 2) within the system 10(FIG. 1) and, optionally, in respective vessels 58 (FIG. 3A) or vesselracks 84 (FIG. 3B) loaded into the system 10 (FIG. 1) consistent withembodiments of the present invention. As illustrated in FIG. 16F, eachvessel 58 (FIG. 3A) or vessel rack 84 (FIG. 3B) is illustrated in aparticular row and, if appropriate, each specimen 23 (FIG. 2) of thatvessel 58 (FIG. 3A) or vessel rack 84 (FIG. 3B) is illustrated in acorresponding column along with some information about that specimen 23(FIG. 2). As such, the specimen location screen 570 may provide the userwith a more easily understood graphical illustration of the location ofany particular specimen 23 (FIG. 2) of the system 10 (FIG. 1) than thespecimen section 552 of FIG. 16E. As an example, the sole sample listedin the specimen section 552 of FIG. 16E is illustrated in FIG. 16F atthe first vessel rack 84 (FIG. 3B) in the first position thereof.Additional control samples are illustrated in the fifth vessel rack 84(FIG. 3B) in corresponding positions. Information corresponding to thesample is also illustrated in the specimen location screen 570,including the ID of the specimen 23 (FIG. 2) (i.e., x234512), as well asthe number of assays that have been performed on prepared samples of thespecimen 23 (FIG. 2) in relation to the total number of assays toperform on prepared samples of the specimen 23 (FIG. 2) (i.e., theindication of “0/2,” with zero being the number of assays that have beenperformed on prepared samples of the specimen 23 (FIG. 2) and two beingthe total number of assays to perform on prepared samples of thespecimen 23 (FIG. 2)).

FIG. 16G illustrates a test status screen 580 that illustrates thestatus of each test to be performed by the system 10 (FIG. 1) consistentwith embodiments of the present invention. Users can sort the tests byassay type with a test type control 582, as well as sort the tests bythose that have specimens 23 (FIG. 2) that are currently loaded in thesystem 10 (FIG. 1), generally, or in a vessel 58 (FIG. 3A), on vesselracks 84 (FIG. 3B) in the system 10 (FIG. 1) with a vessel rack control584. In any event, the test status screen 580 indicates the request typeof each test (e.g., as illustrated, “Patient” requests), the ID of thespecimen 23 (FIG. 2) for each test, and the assay type for the test. Thetest status screen 580 further illustrates the status of each test andany notes associated with the tests, as well as the rack position of thespecimen 23 (FIG. 2) for each test and whether the vessel 58 (FIG. 3A)or the vessel rack 84 (FIG. 3B) with the specimen 23 (FIG. 2) of thetest is loaded into the system 10 (FIG. 1).

A person having ordinary skill in the art will recognize that theenvironments illustrated in FIGS. 1-16G are not intended to limit thescope of embodiments of the present invention. In particular, system 10,the sample preparation station 20, sample preparation controller 22, thesample analysis station 24, and/or the sample analysis controller 26 mayhave alternative configurations consistent with alternative embodimentsof the invention. For example, the sample preparation station 20 and thesample analysis station 24 may not be provided in a single housing butmay instead be physically spaced from each and connected through atleast one data communication link (e.g., such as links 315 a, 315 b, 315c and/or a link through network 30) as well as through at least onetransport mechanism (e.g., to provide prepared samples from the samplepreparation station 20 to the sample analysis station 24). Also forexample, the sample preparation station 20 and the sample analysisstation 24 may be integrated with each other within a single housing,yet retain their respective and distinct functions.

Moreover, the system 10, the sample preparation station 20, the samplepreparation controller 22, the sample analysis station 24, and/or thesample analysis controller 26 may include fewer or additional componentsconsistent with alternative embodiments of the present invention.Indeed, a person having skill in the art will recognize that otheralternative hardware and/or software environments may be used withoutdeparting from the scope of the present invention. For example, thecontrol application 318 and/or the sample analysis application 418 maybe configured with fewer or additional modules, while the memory 304 maybe configured with fewer or additional data structures. Additionally, aperson having ordinary skill in the art will appreciate that the samplepreparation controller 22 and/or sample analysis controller 26 mayinclude more or fewer applications disposed therein. As such, otheralternative hardware and software environments may be used withoutdeparting from the scope of the present invention.

Still further, a person having ordinary skill in the art will recognizethat the screenshots illustrated in FIGS. 16A-16G are not intended tolimit the scope of embodiments of the present invention. In particular,the screens illustrated by the screenshots of FIGS. 16A-16G may includemore or fewer sections, user interfaces, or human perceptible componentsconsistent with embodiments of the present invention.

The routines executed to implement the embodiments of the presentinvention, whether implemented as part of an operating system or aspecific application, component, program, object, module or sequence ofinstructions executed by one or more computing systems or controllerswill be referred to herein as a “sequence of operations,” a “programproduct,” or, more simply, “program code.” The program code typicallycomprises one or more instructions that are resident at various times invarious memory and storage devices in a computing system or controller,and that, when read and executed by one or more processors of thecomputing system or controller, cause that computing system orcontroller to perform the steps necessary to execute steps, elements,and/or blocks embodying the various aspects of the present invention.

While the present invention has and hereinafter will be described in thecontext of fully functioning computing systems and controllers, thoseskilled in the art will appreciate that the various embodiments of thepresent invention are capable of being distributed as a program productin a variety of forms, and that the invention applies equally regardlessof the particular type of computer readable signal bearing media used toactually carry out the distribution. Examples of computer readablesignal bearing media include but are not limited to physical andtangible recordable type media such as volatile and nonvolatile memorydevices, floppy and other removable disks, hard disk drives, opticaldisks (e.g., CD-ROM's, DVD's, etc.), among others.

In addition, various program code described hereinafter may beidentified based upon the application or software component within whichit is implemented in a specific embodiment of the present invention.However, it should be appreciated that any particular programnomenclature that follows is used merely for convenience, and thus theinvention should not be limited to use solely in any specificapplication identified and/or implied by such nomenclature. Furthermore,given the typically endless number of manners in which computer programsmay be organized into routines, procedures, methods, modules, objects,and the like, as well as the various manners in which programfunctionality may be allocated among various software layers that areresident within a typical computer (e.g., operating systems, libraries,APIs, applications, applets, etc.), it should be appreciated that thepresent invention is not limited to the specific organization andallocation of program functionality described herein.

Consistent with embodiments of the present invention, an automatedpreparation and analysis system may be used to automatically prepare aspecimen for a selected test in accordance with a predetermined assay.FIG. 17 is a flowchart 600 illustrating a sequence of operations thatgenerally describes the operational flow for collecting a specimen,preparing a sample of that specimen, and analyzing a prepared sample ofthat specimen consistent with embodiments of the present invention.Initially a test request, order, or procedure prescription is received,such as from a prescribing physician or a laboratory technician, andinput by a user into a hospital or laboratory system (block 602). Auser, for example, may be a medical transcriptionist, phlebotomist, orlaboratory technician that inputs the test request into a LIS system.The test request is transmitted as appropriate to an individual whocollects the appropriate specimen (block 604) in a manner that is wellknown. For example, blood samples may be collected into septum coveredtest tube via a syringe port while environmental samples may becollected using a swab and deposited into a collection vessel includingtransport media.

Once the specimen is in an appropriate specimen container, such as avessel that may be used by the automated preparation and analysissystem, a user may designate an assay for preparation and analysis ofthe specimen, a priority and/or a desired time to return a result of theanalysis for the specimen, and load the specimen (block 606). Then, inaccordance with the procedures set forth in greater detail below, thesystem samples the specimen and generates a prepared sample that issubsequently analyzed (e.g., the analytes of the prepared sample arequantified) in accordance with one or more assays (block 608). Once theanalysis is complete, the system prepares results and provides them in areport in an appropriate human perceptible form for the user, such asthe prescribing physician or laboratory technician (block 610).

The automated sample preparation and analysis system is configured toautomatically prepare specimens with a sample preparation station foranalysis by a sample analysis station. The sample analysis station, inturn, is configured to analyze the prepared sample generated from thespecimen and output data relating to an analysis of the analytes of theprepared sample. The sample preparation station is under control of asample preparation controller while the sample analysis station is underthe control of a sample analysis controller.

FIG. 18 is a flowchart 640 illustrating a sequence of operations for thesample preparation controller to operate the sample preparation stationto prepare a sample consistent with embodiments of the presentinvention. Initially, the sample preparation controller determineswhether a specimen has been loaded to the system (block 642). When aspecimen has not been loaded (“No” branch of decision block 642), thesequence of operations returns back to block 642. When a specimen hasbeen loaded (“Yes” branch of decision block 642) the sample preparationcontroller determines at least one order, i.e., a test, for the sample(block 644) and determines information associated with the at least oneorder, including at least one assay type to perform as well as a TargetTime to Result (hereinafter referred to, and illustrated as, “TTR”) andoptionally a Target Time to Prepare the Sample (hereinafter referred to,and illustrated as “TTP”), for each assay (block 646). In particular,the sample preparation controller may determine the order for testingthe sample and the information associated with that order via a userinput device of the sample preparation controller or by associating anindication of assay included within a barcode or RFID antenna on thevessel with information stored at the sample preparation controllerstored across a network, such on a LIS.

When an assay type and its respective TTR have been determined, thesample preparation controller is configured to select the assay from aplurality of unique assays to be performed on the specimen (block 648),including the methodologies for preparing and analyzing the sample. Forexample, the assay may specify the types of reagents to add to thesample, whether mixing, centrifuging, and/or incubation of the sampleare required, and an indication of the type of analysis to perform onthe prepared sample.

After selecting the assay for each assay type ordered for the specimen(block 648), the sample preparation controller schedules the samplepreparation (block 650). After sample preparation is scheduled (block650), the sample preparation controller controls the sample preparationstation to move a new vessel to a sampling station (block 652). Thesample preparation controller may then determine whether the particularspecimen to be loaded should be mixed (block 653). If the specimen is ofthe type that separation may inherently occur over time, for example,the separation of red blood cells from the plasma in a blood specimen,then mixing may be necessary to ensure a proper sampling of the specimenis loaded for testing. If mixing is necessary, or desired (“Yes” branchof decision block 653), then the specimen is mixed (block 655). Mixingmay occur in any manner that is generally known, includingaspirating/dispensing multiple times with the sample pipette assembly,stirring the specimen with the pipette shaft of the sample pipetteassembly, use of a stir bar, use of a vortex mixer, or any other knownmethod. With mixing complete, or if the determination was that mixingwas not necessary (“No” branch of decision block 653), then at least aportion of the specimen (i.e., a sample of the specimen) is aspiratedand loaded into the vessel (block 654). The sample preparationcontroller may then control the sample preparation station to preparethe sample for analysis, such as by LCMS.

For example, in conventional mass spectrometry of samples for analysisof the presence and quantitation of a small molecule analyte, samplesare prepared by combining them with one or more reagents or solvents ina vessel, mixing the contents of the vessel, then separatingcontaminants from a supernatant containing one or more analytes ofinterest. The assay may define one or more reagents to mix with thesample, if any, at one or more time points in the assay, and when andwhether to mix and/or use the matrix interference removal station withthat sample. As such, the sample preparation controller may optionallyload one or more reagents and/or one or more internal standards (i.e., aknown quantity of an analyte for quantitative comparative analysis) tothe vessel according to the assay (e.g., the assay associated with thesample in the vessel) (block 656), optionally move the vessel to themixing station and mix the contents of that vessel according to theassay (block 658), and optionally move the vessel to the matrixinterference removal station to separate precipitating solids fromresulting supernatant liquid according to the assay (block 660). Afterthe sample preparation steps, the sample preparation controller movesthe vessel to an analysis staging station (block 662). At the analysisstaging station, the prepared sample waits for selection and injectioninto the sample analysis station.

In conventional mass spectrometry analysis of samples for the presenceand quantization of a protein or peptide analyte, the samples may beprepared by combining the sample with one or more reagents or proteasesin a vessel, mixing the contents of the vessel, and then isolating oneor more proteins or peptides of interest. The assay may define one ormore reagents or proteases to mix with the sample, if any, at one ormore time points in the assay, and when and whether to mixed and/orincubate the sample. As such, the sample preparation controller mayoptionally load one or more reagents, proteases, and/or one or moreinternal standards (i.e., a known quantity of a labeled analyte forquantitative comparative analysis) to the vessel according to the assay(e.g., the assay associated with the sample in the vessel), optionallymove the vessel to the mixing station and mix the contents of thatvessel according to the assay, and optionally move the vessel to theincubator according to the assay. After the sample preparation steps,the sample preparation controller moves the vessel to an analysisstaging station. At the analysis staging station, the prepare samplewaits for selection and injection into the sample analysis station.

FIG. 19 is a flowchart 670 illustrating a sequence of operations for thesample preparation controller to operate the sample preparation stationto prepare a calibration and/or control standard consistent withembodiments of the present invention. The sample preparation controllerinitially determines whether a calibration and/or control test isrequired (block 672). When a calibration or control is not required(e.g., a calibration and/or control has not been automatically ormanually added to the system) (“No” branch of decision block 672) thesequence of operations returns to block 672. However, when a calibrationor control is required (“Yes” branch of decision block 672) the sampleprep controller is configured to determine and select the appropriateassay for the calibration or control (blocks 674, 676) from a pluralityof unique assays. The vessel containing the calibration or controlstandard may include a barcode and/or RFID antenna that is read by aninformation acquisition device to indicate the appropriate assay to beselected.

After selecting the assay for the calibration or control (block 676),the sample preparation controller schedules the calibration or control(block 678). The sample preparation controller then controls the samplepreparation station to move a new vessel to the sampling station (block680) and load at least one internal standard (i.e., a known quantity ofcontrol standard for control analysis or a known quantity of acalibration standard for a calibration) to the vessel (block 682). Thesample preparation controller may optionally load one or more reagentsor solvents to the vessel according to the assay for preparing thecalibration or control standard (block 684), optionally move the vesselto the mixing station and mix the contents of that vessel according tothe assay (block 686), and optionally move the vessel to the matrixinterference removal station to separate precipitating solids fromresulting supernatant liquid according to the assay (block 688). Afterthe preparation steps, the sample preparation controller moves thevessel to an analysis staging station (block 690). At the analysisstaging station, the prepared calibration or control vessel waits forselection and injection to the sample analysis station.

During operation, the automated sample preparation and analysis systemis configured to prepare a plurality of samples and prioritize thepreparation of those samples according to a Target Time to Result(“TTR”). This allows the system to dynamically change its operation toprepare and analyze a later received sample before an earlier receivedsample. The TTR may be specified by a user or automatically set by thesystem when there is no user specified time. For example, certainspecimen types, such as blood or saliva, may degrade relatively quickly,while alternative specimen types, such as urine, degrade relativelyslowly.

As such, the system may automatically assign a specimen type TTR tospecimen types that degrade relatively quickly (e.g., the specimen typeTTR being a value that ensures that samples from particular specimentypes are processed before they may degrade to a point where they areunusable) and assign a system TTR to specimen types that degraderelatively slowly (e.g., the system TTR being a time before which thesamples from other specimen types should be processed by the system).Additionally, or alternatively, the system may determine that aparticular specimen is a priority specimen without a user specified TTR.As such, the system may assign an adjusted specimen type TTR (e.g., ifthe specimen type is of the type that degrades relatively quickly), anadjusted system TTR (e.g., if the specimen type is of the type that doesnot degrade relatively quickly), or a predetermined priority TTR to theassays for the priority specimens. In specific embodiments, the adjustedspecimen type TTR may be half the normal specimen type TTR, the adjustedsystem TTR may be half the normal system TTR, and the priority TTR maybe specified by a user or manufacturer of the system.

FIG. 20 is a flowchart 700 illustrating a sequence of operations for thesample preparation controller to associate the preparation of a specimenwith an appropriate TTR consistent with embodiments of the invention.Specifically, the sample preparation controller determines if a user hasspecified a time for the completion of the test, i.e., the preparationand analysis of a sample in accordance with a predetermined assay (block702). When the user has specified a time for the completion of the test(“Yes” branch of decision block 702), the sample preparation controllersets the TTR to that user specified time (block 704) and the sequence ofoperations may end. When the user has not specified a time for thecompletion of the test (“No” branch of decision block 702), the samplepreparation controller determines whether the specimen is a priorityspecimen (block 706). When the specimen is a priority specimen (“Yes”branch of decision block 706) the sample preparation controller sets theTTR to an adjusted specimen type TTR, an adjusted system TTR, or apredetermined priority TTR, whichever is shortest (block 708), and thesequence of operations may end. When the specimen is not a priorityspecimen (“No” branch of decision block 706) the sample preparationcontroller determines whether the specimen is of the type that degradesrelatively quickly (block 710). When the specimen is of the type thatdegrades relatively quickly (“Yes” branch of decision block 710), thesample preparation controller sets the TTR to a specimen type TTR thatcorresponds to the specimen type of the sample (block 712) and thesequence of operations may end. When the specimen is not of the typethat degrades relatively quickly (“No” branch of decision block 710),the sample preparation controller determines whether the assayassociated with the specimen has a predetermined TTR (block 713). Whenthe assay associated with the specimen has a predetermined TTR (“Yes”branch of decision block 713), the sample preparation controller setsthe TTR to the TTR for the assay (block 714). However, when the assayassociated with the specimen does not have a predetermined TTR (“No”branch of decision block 713), the sample preparation controller setsthe TTR to the system TTR (block 715).

In some embodiments, it may be beneficial to set a Target Time toPrepare (“TTP”), such as when a number of tests are in queue and theprepared sample would be considered to be more stable than a specimenawaiting preparation. Accordingly, the sample preparation controller mayspecify a TTP for each specimen which indicates a target time for thepreparation of that specimen. The TTP, however, may be subsequentlyadjusted. For example, and as explained above, some specimens may be ofthe type that degrade relatively quickly. For those specimens, it may beadvantageous to prepare them more quickly. As such, and in an optionalstep, the sample preparation controller sets a TTP for the specimen(block 716) and determines whether to adjust the TTP in accordance withthe specimen type and/or another designation by the user (block 717).When the TTP requires no adjustment (“No” branch of decision block 717),the sequence of operations may end. If an adjustment in TTP is required(“Yes” branch of decision block 717), the adjustment is made inaccordance with a predetermined mathematical formula (block 718) and thesequence of operations may end.

A scheduler of the sample preparation controller may be configured todetermine the order in which to prepare samples within the samplepreparation station, as well as reserve the components thereofaccordingly for the system to prepare samples for subsequent analysiswithin the TTR.

FIG. 21 is a flowchart 720 illustrating a sequence of operations for thescheduler to determine the steps to prepare a sample according to apredetermined assay and correspondingly schedule the components of thesample preparation station consistent with embodiments of the presentinvention. The scheduler initially determines if there are any tests(sample preparation and analysis) that have a Remaining Target Time toResult (hereinafter referred to, and illustrated as, “RTTR”) less than apredetermined threshold (e.g., which may be a time much larger than thattypically required to prepare and analyze a sample, and which may beabout an hour) (block 722). When the scheduler determines that there areno tests with an RTTR less than the predetermined threshold (“No” branchof decision block 722), the scheduler determines whether there is acalibration or control standard that needs to be prepared for acalibration or control of the system, i.e., whether there is an assaywith a pending calibration or control (block 724). If there is nocalibration or control standard that needs to be prepared (“No” branchof decision block 724) the scheduler selects the oldest specimen in thesystem (e.g., the specimen that was loaded earliest) (block 726) andselects the test for that oldest specimen with the smallest RTTR (block728).

Returning to block 724, when there is an assay with a pendingcalibration or control requiring calibration standards or controlstandards to be tested (prepared and analyzed) (“Yes” branch of decisionblock 724), the scheduler selects the calibration or control test withthe smallest RTTR (block 730). Returning to block 722, when there is atest with an RTTR less than the predetermined threshold (“Yes” branch ofdecision block 722), the scheduler determines whether there is acalibration or control test request with an RTTR less than thepredetermined threshold (block 732). When there is a calibration orcontrol test with an RTTR less than the predetermined threshold (“Yes”branch of decision block 732), the scheduler selects the calibration orcontrol test with the smallest RTTR (block 730). However, when there isno calibration or control test with an RTTR less than the predeterminedthreshold (“No” branch of decision block 732), the scheduler selects thetest (for a specimen) with the smallest RTTR (block 736).

In response to selecting the test of the oldest specimen with thesmallest RTTR (block 728), selecting the calibration or control testwith the smallest RTTR (block 730), or selecting the test with thesmallest RTTR (block 736), the scheduler determines the preparationsteps and methodologies corresponding to the selected assay (block 738),determines an estimated time required for the test, i.e., thepreparation and analysis steps (e.g., with such a determination takinginto account previously determined estimations for the time required toprepare and/or analyze previous samples) (block 740), and reservessample preparation station components to prepare the sample within thevessel in accordance with the assay and as appropriate with respect tothe RTTR (block 742), if able. In this manner, the scheduler may preparesamples from the specimens or vessels for calibration or control testsappropriately and with respect to their RTTR for analysis within theRTTR.

The sample preparation controller is configured to routinely checkwhether a particular assay may be performed by the system. For example,a particular assay cannot be performed when there is no validcalibration data for that assay or when there is not enough of aparticular consumable (e.g., a reagent, solutions, internal standard,and so forth) required to complete a test in accordance with that assay.For example, the system may be configured to perform categories ofassays, such as therapeutic drug monitoring assays (detection ofimmunosuppressants such as tacrolimus, everolimus, sirolimus, andcyclosporin A or chemotherapeutics such as methotrexate, busulfan,5-fluorouracil, and docetaxel), endocrinological assays (detection of25OH vitamin D2, 25OH vitamin D3, testosterone, cortisol,hydrocortisone, cortisone, progesterone, hydroxyprogesterone, predisone,and androstenedione), and pain management or drugs-of-abuse assays(detection of phencyclidine, benzoylecgonine, cocaine, delta9-THC,11-norDelta, 9-THC-COON, amphetamine, methamphetamine, MDMA,opiates/opiods, hydromorphone, norhydrocodone, norcodeine, morphine,hydrocodone, codeine, noroxycodone, oxymorphone, dihydrocodeine,oxycodone, 6-MAM, tapentadol, norfentanyl, fentanyl, tramadol,methadone, and metoprolol). It would be appreciated that this listing ofpossible analytes is not all-inclusive but rather additional analytes,such as proteins and other large molecules, obtained from patientsamples or environmental samples, may also be analyzed with selection ofproper separation and analytical techniques known to those of ordinaryskill in the art. Each category of assay, in turn, may includeindividual types of assays that are specific to a particular therapeuticdrug, hormone, or drug-of-abuse, and so forth and thus require specificconsumables and/or parameters for preparation and/or analysis thereof.

FIG. 22 is a flowchart 750 illustrating a sequence of operations for thesample preparation controller to determine whether a particular assaymay be performed by the system consistent with embodiments of thepresent invention. For example, the sample preparation controller maydetermine, upon initialization, what types of assays can be performed byinquiring for that information from the sample analysis controller. Thesample preparation controller, in turn, determines whether those typesof assays may be performed. Thus, the sample preparation controllerdetermines whether all types of assays that may be performed by thesystem have been checked (block 752). When all types of assays have notbeen checked (“No” branch of decision block 752) the sample preparationcontroller may select the next assay type (block 754) and determinewhether there are any orders for that selected assay type (block 756).When there are no orders for the selected assay type (“No” branch ofdecision block 756), the sequence of operations may return to block 752.However, when there are orders for the selected assay type (“Yes” branchof decision block 756), the sample preparation controller determineswhether there are sufficient consumables for testing a sample inaccordance with the selected assay type (block 758).

The system may include one or more reagents, solvents, or internalstandards for testing one or more samples in accordance with one or moreassays. The sample preparation controller, in turn, tracks the levels ofeach reagent, solvent, and internal standard and/or the use of eachreagent, solvent, and/or internal standard to determine when suchreagents, solvents, or internal standards are low and/or depleted. Assuch, when the volume of at least one reagent, solvent, and/or internalstandard for use in accordance with the selected assay type isinsufficient (e.g., is low or has a volume that is not enough to use tofully test a sample associated with that selected assay protocol) (“No”branch of decision block 758), the sample preparation controller sets anassay consumables flag (block 760). However, when the volume of eachreagent, solvent, and/or internal standard associated with of theselected assay type is sufficient (“Yes” branch of decision block 758)the sample preparation controller clears the assay consumables flag(block 762).

In response to setting the assay consumables flag (block 760) orclearing the assay consumables flag (block 762), the sample preparationcontroller performs a calibration checking subroutine (block 764) todetermine whether the selected assay type is associated with validcalibration data. After the calibration checking subroutine (block 764)the sample preparation controller determines the status of the selectedassay type (e.g., whether it is time for a calibration assay for theselected assay type or whether the results of a check of the selectedassay type indicate that the present calibration is no longer valid)(block 766). When the status of the selected assay type is not OK (e.g.,the selected assay type is not associated with valid calibration dataand there is a calibration or control test pending for that selectedassay type) (“NOT OK” branch of decision block 766), the samplepreparation controller indicates that the selected assay type is invalidand stores an indication of such in an invalid assay list (block 768).However, when the status of the selected assay type is OK (e.g., theselected assay type is associated with valid calibration data and thereis no calibration or control test pending for that selected assay type)(“OK” branch of decision block 766), the sample preparation controllerdetermines whether the assay consumables flag is set (block 770).

When the assay consumable flag is set (e.g., indicating that the volumeof at least one reagent, solvent, and/or internal standard for use inaccordance with the selected assay type is insufficient) (“Yes” branchof decision block 770), the sequence of operations proceeds to block768. However, when the assay consumable flag is not set (e.g.,indicating that the volume of each reagent, solvent, and/or internalstandard for use in accordance with the selected assay type issufficient) (“No” branch of decision block 770), the sample preparationcontroller indicates that the selected assay type is cleared to be runby the system (e.g., valid) and stores an indication of such in a validassay list (block 772). Returning to block 766, when the status of theselected assay type indicates that a calibration or control test ispending (“CALIBRATION OR QC PENDING” branch of decision block 766), thesample preparation controller determines whether the assay consumablesflag is set (block 774). When the assay consumables flag is not set(“No” branch of decision block 774), the sample preparation controllerindicates that the selected assay type is pending a calibration orcontrol test and stores and indication of such in a pending assay list(block 776) and the sequence of operations may return to block 752.Otherwise, if a consumables flag is set (“Yes” branch of decision block774), then the sequence of operations goes to block 768.

In response to block 768, block 772, or block 776, or in response todetermining that the assay consumable flag is not set for a selectedassay type whose status is that a calibration or control test is pending(“No” branch of decision block 774), the sequence of operations returnsto block 752. Referring to block 752, when all types of assays have beenchecked (“Yes” branch of decision block 752), the sample preparationcontroller outputs and/or stores information about each assay type,including their statuses and whether there are any missing or lowconsumables (block 778), after which the sequence of operations may end.

FIG. 23 is a flowchart 780 illustrating a sequence of operations for thesample preparation controller to perform a calibration checkingsubroutine consistent with embodiments of the present invention.Initially, the sample preparation controller determines whether there iscalibration data for the selected assay type (block 782). When there isno calibration data for the selected assay type (“No” branch of decisionblock 782), the status of the selected assay type is set to “NOT OK”(block 784) and the sequence of operations may end. However, when thereis calibration data for the selected assay type (“Yes” branch ofdecision block 782), the sample preparation controller determineswhether the calibration data for the selected assay type has beenaccepted by the user as an appropriate mathematical model for relativequantification of the selected assay type with one or more known controlsamples (block 786). When the user has accepted the calibration data(“Yes” branch of decision block 786), the status of the selected assaytype is set to “OK” (block 788) and the sequence of operations may end.

However, when the user has not accepted the calibration data (“No”branch of decision block 786), the sample preparation controllerdetermines whether the user has entered calibration coefficients suchthat actual calibration assays for the selected sample type are notnecessary (e.g., whether the calibration is “constant”) (block 790).When the calibration is constant (“Yes” branch of decision block 790),the sequence of operations may proceed to block 788. However, when thecalibration is not constant (“No” branch of decision block 790), thesample preparation controller determines whether there is a calibrationor control test for the selected assay type pending (block 792). Whenthere is not a calibration or control test for the selected assay typepending (“No” branch of decision block 792), the sequence of operationsmay proceed to block 784. However, when there is a calibration orcontrol test for the selected assay type pending (“Yes” branch ofdecision block 792), the status of the selected assay type is set to“PENDING” (block 794) and the sequence of operations may end.

During operation, the sample preparation controller is configured toselect a particular assay to perform based on information generated bythe scheduler. However, factors may arise that force the samplepreparation controller to deviate from a previous schedule, includingthe scheduling of calibration tests, the scheduling or need for QCtests, and/or priority tests that have been input after, but performedbefore, the particular test.

The sample preparation controller is thus configured to determinewhether a test can be performed in accordance with a particular assaywhen that assay is selected to be performed.

FIG. 24 is a flowchart 800 illustrating a sequence of operations for thesample preparation controller to perform or override a particular testconsistent with embodiments of the invention. The sample preparationcontroller initially determines whether there is a free vessel positionto receive a vessel (e.g., such as a vessel that will contain the sampleand/or consumables appropriate for the test) (block 802). When there isno free vessel position (“No branch of decision block 802), the samplepreparation controller may cancel the sample preparation stationcomponent reservations for the test (block 804) and the sequence ofoperations may end. However, when there is a free vessel position (“Yes”branch of decision block 802), the sample preparation controllerdetermines whether a calibration test is pending for the assay type(block 806).

When a calibration test is pending for the assay type (“Yes” branch ofdecision block 806), the sample preparation controller may override thesample test (“test”) with a calibration test for the assay type (block808). When there is no calibration test pending for the assay type (“No”branch of decision block 806), the sample preparation controllerdetermines whether the number of runs for the assay type are greaterthan or equal to a stability check indicator, or whether there is acontrol test scheduled for the assay type (block 810).

A control test may be periodically performed for a check of the validityof the current calibration data for the assay type. In one particularembodiment, the control test may be performed about every twenty runs ofits respective assay type, or after a defined run time, or “QC time.” Assuch, the sample preparation controller may increment a run indicatoreach time a test is completed in accordance with a particular assay typeto track the number of runs of the particular assay type. Alternatively,a user may manually schedule a control test. Thus, when the runindicator for the assay type is not greater than or equal to thestability check indictor, or when the user has not manually scheduled acontrol test for the assay type (“No” branch of decision block 810), thesample preparation controller increments the run indicator for the assaytype (block 812).

However, when the run indicator for the assay type is greater than orequal to the stability check indicator, a defined control time hasexpired, or when the user has manually scheduled a control test for theassay type (“Yes” branch of decision block 810), the sample preparationcontroller clears the run indicator (block 814) and determines whetherthe volumes of the reagents, solvents, and/or internal standards used bya control test associated with the pending assay is sufficient (block816). When the volume of at least one reagent, solvent, and/or internalstandard used by the control test associated with the pending assay isinsufficient (“No” branch of decision block 816), the sample preparationcontroller indicates and/or stores data indicating that there is atleast one insufficient consumable for the control test associated withthe pending assay (block 818) and proceeds to block 804. However, whenthe volume of each reagent, solvent, and/or internal standard for thecontrol test is sufficient for that assay (“Yes” branch of decisionblock 816), the sample preparation controller may override the test witha control test for the assay type (block 820).

In response to overriding the test with the calibration test (block808), incrementing the run indicator for the assay type (block 812), oroverriding the test with the control test (block 820), the samplepreparation controller determines whether the sample preparation stationcomponent reservations for the test, calibration test, or control testcomport with the availability of those components (e.g., whether thepreparation for the sample may still be performed by the components ofthe sample preparation station with respect to previous reservationstherefor) (block 822). When the sample preparation station componentreservations no longer comport with the availability of those components(“No” branch of decision block 822), the sequence of operations proceedsto block 804. However, when the sample preparation station componentreservations still comport with the availability of those components(“Yes” branch of decision block 822) the sample preparation controllerperforms the sample preparation (e.g., whether it is the original testfor the sample, the test for the calibration standard that overrode thatoriginal test, or the control standard that overrode that original test)(block 824).

The sample preparation controller is configured to monitor thecomponents of the sample preparation station to effectively prepare thesample in the system. For example, when the sample preparation stationincludes a centrifuge and mixer, the sample preparation controller isconfigured to monitor the operation and loading of those components tomaximize the number of vessels processed thereby. More specifically, thesample preparation controller may be configured to monitor which vesselsrequire processing by a mixer, which vessels require processing by acentrifuge, and control the loading and operation of the mixer andcentrifuge to operate on those vessels appropriately.

FIG. 25 is a flowchart 830 illustrating a sequence of operations for thesample preparation controller to monitor the operation of the centrifugeand the mixer consistent with embodiments of the present invention. Thesample preparation controller may determine whether the centrifuge isavailable within a predetermined number of seconds, which, for example,may be about sixty seconds (block 832). When the centrifuge is availablewithin the predetermined number of seconds (“Yes” branch of decisionblock 832), the sample preparation controller determines whether thereare vessels associated with a test having an assay that calls for thenext preparation step for those vessels to be performed with acentrifuge (e.g., vessels to be centrifuged, such as vessels that havealready been processed by the mixer) (block 834). When there are vesselsto be centrifuged (“Yes” branch of decision block 834), the samplepreparation controller may optionally operate the sample preparationstation to load at least one vessel rack with the vessels to becentrifuged (block 836) and determines whether there are full vesselracks for processing with the centrifuge (block 838). When there are notfull vessel racks for processing with the centrifuge (“No” branch ofdecision block 838), the sample preparation controller determineswhether all vessels have been prepared (block 840).

Otherwise, if vessel racks are not used, then when there are vessels tobe centrifuged (“Yes” branch of decision block 834), then the samplepreparation controller operates the sample preparation station to loadtwo or more vessels into the centrifuge. When there are not sufficientvessels to fill the available positions in the centrifuge (“No” branchof decision block 838), the sample preparation controller determineswhether all vessels have been prepared (block 840).

In any event, when there are sufficient vessels to fill the availablepositions in the centrifuge (“Yes” branch of decision block 838) and/orwhen all vessels have been prepared (“Yes” branch of decision block840), the sample preparation controller loads the centrifuge with theappropriate vessels and centrifuges the vessels (block 842).

When the centrifuge is not available within the predetermined number ofseconds (“No” branch of decision block 832), when there are no vesselsassociated with a test having an assay that calls for the nextpreparation step for those vessels to be performed with a centrifuge(“No” branch of decision block 834), or when all vessels have not beenprepared (“No” branch of decision block 840), the sample preparationcontroller determines whether the mixer is available within apredetermined number of seconds, which, for example, may also be aboutsixty seconds (block 844). When the mixer is not available within thepredetermined number of seconds (“No” branch of decision block 844), thesequence of operations may return to block 832. However, when the mixeris available within the predetermined number of seconds (“Yes” branch ofdecision block 844), the sample preparation controller determineswhether there are vessels associated with a test associated with anassay that calls for the next preparation step for those vessels to beperformed with a mixer (e.g., vessels to be mixed) (block 846).

When there are no vessels to be mixed (“No” branch of decision block846), the sequence of operations may return to block 832. However, whenthere are vessels to be mixed (“Yes” branch of decision block 846), thesample preparation controller may optionally load the vessel racks, ifused, (block 848) and determines whether there are sufficient vesselsfor processing with the mixer (block 850). When there are not sufficientvessels for processing with the mixer (“No” branch of decision block850), the sample preparation controller determines whether all vesselshave been prepared (block 852). Thus, when there are sufficient vesselsfor processing with the mixer (“Yes” branch of decision block 850) orwhen all vessels have been prepared (“Yes” branch of decision block852), the sample preparation controller loads the mixer with theappropriate number of vessels and operates on the vessels to mix thecontents thereof (block 854). However, when all vessels have not beenprepared (“No” branch of decision block 852), the sequence of operationsmay return to block 832.

In some embodiments, the sample preparation controller may be configuredto determine to load vessels to the centrifuge or mixer, without regardto whether all the centrifuge positions are filled with vessels orwhether all vessels have been prepared. As such, the sample preparationcontroller may analyze the RTTRs of the tests associated with eachvessel to determine whether the smallest RTTR is less than acentrifuging threshold to determine whether to load and run thecentrifuge and/or whether the smallest RTTR is less than a mixingthreshold to determine whether to load and run the mixer. As such, ifthere is a sample associated with a small RTTR, such as a prioritysample, the centrifuge and/or the mixer may not be loaded to fullcapacity despite there being one or more specimens, samples, and/orvessels that still require processing. The sample preparation controllermay be configured to determine availability and schedule vessels forsample preparation components other than or in addition to the exemplarycentrifuge and mixer, for example, a matrix interference removal stationor an incubator.

FIGS. 26A-26C are a flowchart 860 illustrating a sequence of operationsfor the sample preparation controller to control the sample preparationstation and sample analysis station to load a prepared sample, fill asample loop of an injection valve, and perform housekeeping operationson the vessel, the injection port, and the injection valve consistentwith embodiments of the present invention. In particular, the flowchart860 is performed with respect to tests that are associated with preparedsamples. The sample preparation controller begins by determining, basedon port availability data sent to the sample preparation controller fromthe sample analysis controller via a high level data interface link(i.e., a TCP/IP link) (block 862), whether a port is available in Nseconds (e.g., which may correspond to the time required to move avessel to the rotatable table, then aspirate and dispense the preparedsample in that vessel to an injection port) (block 864). When theinjection port is not available in N seconds (“No” branch of decisionblock 864) the sequence of operations may return to block 864. However,when the injection port is available in N seconds (“Yes” branch ofdecision block 864), the sample preparation controller determineswhether there are any tests with an RTTR less than a predeterminedthreshold (block 866). When there is no test with an RTTR less than thepredetermine threshold (“No” branch of decision block 866), the samplepreparation controller determines whether there is a pending calibrationor control test (block 868). When there is not a pending calibration orcontrol test (“No” branch of decision block 868), the sample preparationcontroller selects the test with the smallest RTTR that matches thecurrent assay type being performed by the sample analysis station (block870). When there is a pending calibration or control test (“Yes” branchof decision block 868), the sample preparation controller selects thecalibration or control test with the smallest RTTR (block 872).

Returning to block 866, when there is a test with an RTTR less than thepredetermined threshold (“Yes” branch of decision block 866), the samplepreparation controller determines whether there is a calibration orcontrol test with an RTTR less than the predetermined threshold (block874). When there is a calibration or control test with an RTTR less thanthe predetermined threshold (“Yes” branch of decision block 874), thesample preparation controller selects the calibration or control testwith the smallest RTTR (block 876). However, when there is nocalibration or control test with an RTTR less than the predeterminedthreshold (“No” branch of decision block 874), the sample preparationcontroller determines whether there is a test with an RTTR less than acritical time (e.g., which may correspond to a typical time required forswitching the chemistries associated with the LC columns, such as one ormore stationary or mobile phases) (block 878).

When there is a test with an RTTR less than the critical time (“Yes”branch of decision block 878), the sample preparation controller selectsthe test with the smallest RTTR (block 880). When there is no test withan RTTR less than the critical time (“No” branch of decision block 878),the sample preparation controller determines whether there is a testthat matches the current assay type that is being performed by thesample analysis station (block 882). When there is no test that matchesthe current assay type that is being performed by the sample analysisstation (“No” branch of decision block 882), the sequence of operationsproceeds to block 880.

However, when there is a test that matches the current assay type thatis being performed by the sample analysis station (“Yes” branch ofdecision block 882), the sample controller selects the test that matchesthe current assay type that is being performed by the sample analysisstation and that has the smallest RTTR (block 884). In response toselecting the test (blocks 870, 872, 876, 880, or 884), the samplepreparation station reserves the sample analysis station components toperform the test in accordance with the assay (block 886) and moves thevessel for the selected test to the rotatable table (block 888). Thesample preparation controller than transmits data associated with theselected assay to the sample analysis controller (block 890). Such datais sent from the sample preparation controller to the sample analysiscontroller via the high level data interface link (block 892) and mayinclude an indication of the test to perform, what analytes to detectwith the assay type, the amount of prepared sample for the test, and aGUID of the sample.

Once data associated with the test has been transmitted to the sampleanalysis controller (block 890), the sample controller operates theinjector pipette assembly to aspirate the prepared sample from thevessel associated with that test (e.g., the vessel in the rotatabletable) (block 894) and determines whether the loop intended for theprepared sample is available to accept the prepared sample (block 896)based on data from the sample analysis controller transmitted via thelow level data link indicating same (block 898). When the samplepreparation controller determines that the loop intended for theprepared sample is not available (“No” branch of decision block 896) thesequence of operations may return to block 896. However, when the samplepreparation controller determines that the loop intended for theprepared sample is available (“Yes” branch of decision block 896), thesample preparation controller transmits a command (block 900) to thesample analysis controller via the low level data link to switch thevalve intended for the prepared sample to a “fill in loop” position(e.g., such as that illustrated in FIG. 10B) (block 902).

The sample preparation controller may also determine whether the valvehas been switched (block 904) based on data from the sample analysiscontroller transmitted via the low level data link indicating the same(block 906). When the sample preparation controller determines that thevalve has not been switched (“No” branch of decision block 904) thesequence of operations may return to block 904. When the samplepreparation controller determines that the valve has been switched(“Yes” branch of decision block 904) the sample preparation controllertransmits a command (block 908) to the sample analysis controller viathe low level data link to fill the loop (block 910) and moves theinjector pipette assembly with the prepared sample to the injector portintended for the prepared sample and dispenses the prepared sample tothat injector port (block 912).

While dispensing the sample to the injection port, the samplepreparation controller determines whether sufficient prepared sample hasbeen dispensed (e.g., which may be a volume of prepared sample havingthree times the volume of the loop to sufficiently flush the loop withthe prepared sample) (block 914) based on data from the sample analysiscontroller transmitted via the low level data link indicating same(e.g., that the injection port is “ready”) (block 916). When there hasnot been sufficient prepared sample dispensed (“No” branch of decisionblock 914), the sample preparation controller operates the injectorpipette assembly to dispense more prepared sample to the injection portand returns to block 914. However, when there has been sufficientprepared sample dispensed (“Yes” branch of decision block 914), thesample preparation controller stops the injector pipette assembly fromdispensing the prepared sample and transmits a command (block 918) toswitch the valve to the “in-line” position to flush the loop transmittedvia the low level data link (block 920). The sample preparationcontroller then determines whether the valve has been switched (block922) based on data from the sample analysis controller transmitted viathe low level data link indicating the same (block 924). When the valvehas not been switched (“No” branch of decision block 922), the sequenceof operations may return to block 922. However, when the valve has beenswitched (“Yes” branch of decision block 922), the sample preparationcontroller moves the remaining prepared sample to waste, washes theinjector pipette assembly and the injection port (including fluid linesand internal channels of the valve, as appropriate) (block 926), andproceeds back to block 864.

FIGS. 27A-27B are a flowchart 930 illustrating a sequence of operationsfor the sample analysis controller to determine an assay type to performand operate the sample analysis station to switch a valve to a “fill inloop” position, then switch the valve to an “in-line” positionconsistent with embodiments of the present invention. Specifically, thesample analysis controller transmits injection port availability data(block 932) indicating the availability of at least one injection portof at least one respective LC channel of the LC station via the highlevel data interface link (block 934). In one embodiment, the sampleanalysis controller transmits the injection port availability data foran injection port when that respective injection port will be availablein N seconds. In an alternative embodiment, the sample analysiscontroller periodically batch transmits injection port availability datafor all the injection ports of the LC station such that the samplepreparation controller separately tracks the availability of thoseinjection ports. In a further alternative embodiment, the sampleanalysis controller continuously updates injection port availabilitydata for all the injection ports of the LC station. In any event, thesample analysis controller determines whether it has received dataassociated with the assay (block 936) based on data transmitted by thesample preparation controller to the sample analysis controller over thehigh level data interface link (block 938). When the sample analysiscontroller has not received data associated with the assay (“No” branchof decision block 936), the sequence of operations may return to block936. However, when the sample analysis controller has received dataassociated with the assay (“Yes” branch of decision block 936), thesample analysis controller may determine, from that data, the assay typeto perform and the methodology to use to perform the test, including butnot limited to the analytes of interest to analyze (block 940).

After determining the assay type to perform and the methodology therefor(block 940), the sample analysis controller prepares the sample analysisstation for that assay type (block 942). For example, the preparation ofthe sample analysis station for the assay type may include determiningwhich of the LC channels of the LC station will receive the nextprepared sample and determining operational parameters of the LC columnsand/or the mass spectrometer. These operational parameters, in turn, mayinclude determining the mobile phase buffer solution flow rate, thecomposition of the mobile phase buffer solution, the ratio of an aqueousmobile phase buffer solution to a non-aqueous mobile phase buffersolution, a gradient for varying the ratios of the aqueous andnon-aqueous mobile phase buffer solutions, an ionization voltage, adesolvation temperature, a lens amplitude, a collision gas temperature,and a collision gas pressure, to name a few. The sample analysiscontroller also determines whether the loop of the valve that is toaccept the prepared sample associated with the test is available toaccept a prepared sample (block 944). When the loop of the valve is notavailable to accept the prepared sample (“No” branch of decision block944), the sequence of operations may return to block 944.

However, when the loop of the valve is available to accept the preparedsample (“Yes” branch of decision block 944), the sample analysiscontroller transmits an indication (block 946) that the loop isavailable to accept the prepared sample via the low level data link(block 948) and the sample analysis controller determines whether toswitch the valve to the “fill in loop” position (block 950) based on acommand to switch the valve to the “fill in loop” position transmittedvia the low level data link (block 952). When the sample analysiscontroller determines not to switch the valve to the “fill in loop”position (“No” branch of decision block 950) the sequence of operationsmay return to block 950. However, when the sample analysis controllerdetermines to switch the valve to the “fill in loop” position (“Yes”branch of decision block 950) the sample analysis controller switchesthe valve to the “fill in loop” position (block 954) and determineswhether to pressurize the loop (block 956) based on a command to fillthe loop transmitted via the low level data link (block 958). When thesample analysis controller determines not to fill the loop (“No” branchof decision block 956), the sequence of operations may return to block956. However, when the sample analysis controller determines to fill theloop (“Yes” branch of decision block 956), the sample analysiscontroller fills the loop (block 960) with the prepared sample.

After filling the loop (block 960), the sample analysis controllerdetermines whether there has been sufficient prepared sample dispensedto the injection port (block 962). When there has not been sufficientprepared sample dispensed to the injection port (“No” branch of decisionblock 962), the sequence of operations may return to block 962. However,when there has been sufficient prepared sample dispensed to theinjection port (“Yes” branch of decision block 962), the sample analysiscontroller transmits an indication (block 964) that the injector port isready via the low level data link (block 966) and determines whether toswitch the valve to the “in-line” position (block 968) based on acommand transmitted from the sample preparation controller via the lowlevel data link (block 970). When the sample analysis controllerdetermines that the valve should not be switched (“No” branch ofdecision block 968) the sequence of operations may return to block 968.However, when the sample analysis controller determines that the valveshould be switched (“Yes” branch of decision block 968), the sampleanalysis controller switches the valve to the “in-line” position,thereby pressurizing the loop, and transmits an indication (block 972)as such via the low level data link (block 974).

Various modules of the sample analysis application of the sampleanalysis controller are configured to operate in a modular fashion. Forexample, the sample analysis station service module and the acquisitionservice module are configured to share data therebetween to operate thesample analysis station.

FIG. 28 is a flowchart 980 illustrating a sequence of operations for asample analysis station service module and/or data manager of a sampleanalysis controller to build a methodology for the analysis and dispatchthat test for performance by the sample analysis station consistent withembodiments of the present invention. In particular, the sample analysisstation service module is configured to receive data associated with thetest that may include an indication of the assay type to perform, whatanalytes to detect, the amount of prepared sample for the test, and aGUID of the sample (block 982). In turn, the data manager may analyzethat received data and determine an assay type to perform from thatreceived data (block 984).

Once the data manager has determined the assay type (block 984) itbuilds the methodology and parameters for the sample analysis station toperform the analysis of the determined assay (block 986) based on dataassociated with that assay type in a configuration data structure file(e.g., an XML configuration data file) (block 988). Moreover, whenbuilding the methodology, the data manager may validate that thecomponents of the sample analysis station are capable of performing thetest (block 986). Validation of the components may include determiningwhether the components of the sample analysis station are able toperform their individual portions of the methodology for the test aswell as reserving the components of the sample analysis station toperform their portions of the methodology for the test.

To store results of the test, the data manager also generates datastructures required to implement the methodology for the test orrequired to store results (block 990). For example, the data manager maycreate data files and/or data folders that are used to store theresults. In any event, the sample analysis station service modulesubmits the methodology for the assay type and any parameters to theacquisition service module for that acquisition service module tooperate the sample analysis station and perform the test (block 992).After submitting the methodology for the test (block 992), the sampleanalysis station service module and/or data manager monitor forindications of events and an indication that the analysis has completed(block 994). When the sample analysis station service module or datamanager determines that an event has occurred (e.g., such as errors orstatus changes) (“Event” branch of decision block 994), the sampleanalysis station service module or data manager logs that event andtransmits that event to the sample preparation controller for thatsample preparation controller to notify a user (block 996).

However, when the sample analysis station service module or data managerdetermines that the test is complete (“Complete” branch of decisionblock 994), the sample analysis station service module determineswhether any post-processing of the raw results data is required andperforms that post-processing, if applicable (block 998). Thedetermination of whether post-processing is required and whatpost-processing to perform, if any, is determined based on theconfiguration data structure file (block 988). For example, the rawresult data may include total ion count (“TIC”) measured at the iondetector of the mass spectrometer. This TIC may include measurements ofthe ion counts for one or more analytes, some of which may be of nodiagnostic interest for the current test. As such, the sample analysisstation service module may post-process the raw result data by filteringthe ion data for one or more analytes from the total ion data. In turn,the sample analysis station service module stores and transmits resultsdata to the sample preparation controller (block 1000).

As described in detail above, the acquisition service of the sampleanalysis application is configured to interface with the components ofthe sample analysis station through various virtual interfaces. Theacquisition service and virtual interfaces, in turn, follow themethodology for the assay determined from the sample analysis stationservice module or data manager.

FIG. 29 is a flowchart 1010 illustrating a sequence of operations forthe acquisition service and the virtual interfaces to implement the testand collect data for the appropriate analyte consistent with embodimentsof the present invention. Upon initialization (“Yes” branch of decisionblock 1012), the acquisition service determines what virtual interfacesare configured for the sample analysis station and initializes thosevirtual interfaces (block 1014). The acquisition service determineswhich virtual interfaces are configured for the sample analysis stationbased on a registered virtual interface configurations data file (block1016). The acquisition service also determines whether a methodology forthe test (block 1018) has been received from the sample analysis stationservice module and/or the data manager (block 1020). When a methodologyhas not been received (“No” branch of decision block 1020), the sequenceof operations may return to block 1020. When a methodology has beenreceived (“Yes” branch of decision block 1020), the acquisition servicenotifies virtual interfaces of the test (block 1022). In turn, the MUXVI determines the order in which the tests are to be performed by thesample analysis station (e.g., when the sample analysis station iscapable of performing multiple tests in a serial or multiplexed fashion)and determines the appropriate LC-channel to receive the prepared sample(block 1024).

In response to the virtual interfaces indicating that they are ready torun the test, and in particular to the MUX VI indicating that it isready to run the test (block 1026), the acquisition service revalidatesthe test with the components of the sample analysis station (e.g., thatthe components of the sample analysis station are able to perform theirindividual portions of the assay as well as that the reservations of thecomponents are still valid) (block 1028) and downloads the methodologyfor the assay, as well as any operational parameters, to the componentsof the sample analysis station (block 1030). In particular, this mayinclude collecting one or more analytes from the LC and dispensing theanalytes to the ionization source (block 1032), then ionizing theanalytes (block 1034). The acquisition service then determines whetherto acquire data for the ionized analytes (block 1036).

In particular, the actual window of interest to monitor for elutedanalytes of interest may be only a portion of the total time requiredfor the test. As such, the acquisition service may be configured tomonitor for selected analytes during an appropriate retention timewindow. Thus, when the acquisition service determines that it is at theappropriate retention time window in which to collect data for one ormore analytes of interest (“Yes” branch of decision block 1036), theacquisition service acquires the data and stores it in the files and/orfolders that were previously set up by the sample analysis stationservice module and/or the data manager (block 1038). When theacquisition service determines that it is not at the appropriateretention time window (“No” branch of decision block 1036), the sequenceof operations may return to block 1036. In any event, when dataacquisition is not complete (e.g., the retention time window for one ormore analytes of interest has not passed) (“No” branch of decision block1040), the acquisition service continues to acquire data and thesequence of operations may return to block 1038. Thus, when dataacquisition is complete (e.g., the retention time window is over or haspassed) (“Yes” branch of decision block 1040), the acquisition servicesends the acquired data to the sample analysis station service module(block 1042) and the sequence of operations may end. According toanother embodiment, the acquisition service may monitor for elutedanalytes, including but not limited to specific analytes of interest,during the entire sample elution time.

FIG. 30 is a flowchart 1050 illustrating a sequence of operations forthe sample preparation controller to maintain and monitor the operationof the sample analysis station consistent with embodiments of thepresent invention. The sample preparation controller may determinewhether the mass spectrometer is available within a predetermined numberof minutes for receiving a prepared sample from the injector pipetteassembly, which, for example, may be a range from about two minutes toabout six minutes (block 1052). When the mass spectrometer is availablewithin the predetermined number of minutes, (“Yes” branch of decisionblock 1052), the sample preparation controller determines whether thereis a prepared sample ready for analysis (block 1054). If a preparedsample is ready for analysis (“Yes” branch of decision block 1054), thenthe sample preparation controller will continue in a manner to determinehow to inject the prepared sample into the sample analysis station.Otherwise, if there is no prepared sample ready to undergo analysiswithin the sample analysis station (“No” branch of decision block 1054)or the mass spectrometer is not available (“No” branch of decision block1052), then the sequence returns to block 1052.

In some embodiments of the invention, the automated sample preparationand analysis system is configured to analyze a plurality of preparedsamples in a multiplexed fashion. According to at least one of thoseembodiments, and after the sample preparation controller has determinedthat a prepared sample is ready for analysis, the MUX VI may determinewhether the first LC channel is available to receive the prepared sample(block 1056), which may consider factors such as whether the first LCchannel has eluted all of an earlier prepared sample, whether theappropriate columns are in-place within the LC channel and ready toaccept the prepared sample in accordance with the test, whether theappropriate mobile phase is in use in accordance the test, and whetherthe flow rate of the mobile phase through the first LC channel isappropriate in accordance with the test. If the determination results inthe first LC channel being available (“Yes” branch of decision block1056), then the first LC channel injection port is available (block1058). However, if the determination results in the first LC channel notbeing available (“No” branch of decision block 1056), then the MUX VIwill determine whether the second LC channel is available to receive theprepared sample (block 1060), which may consider factors that aresimilar to those that were described with reference to the decision ofthe first LC channel availability. If the second LC channel is available(“Yes” branch of decision block 1060), then the second LC channelinjection port is available (block 1062). When the second LC channel isnot available (“No” branch of decision block 1060), the sequence ofoperation returns the determination of mass spectrometer availability(block 1052). In any event, when the MUX VI determines whether aparticular LC channel is available it may send an indication of such tothe sample preparation controller through the sample analysis stationservice module. In some embodiments, the MUX VI determines theavailability of LC channels proactively (e.g., for a particular time inthe future) and provides the sample preparation controller withinjection port availability data corresponding to the futureavailability of injection ports that corresponds to the futureavailability of respective LC channels.

With the appropriate LC channel availability determined, the samplepreparation controller determines the retention times of the one or moreanalytes within the prepared sample under consideration and uses thisdetermination to reserve the mass spectrometer for a time correlatingthe retention time of the analytes in order to measure the analyteeluted at the appropriate retention time window (block 1064). The samplepreparation controller will then generate injection port availabilitydata for the appropriate injection port for the injector pipetteassembly to dispense an aliquot of the prepared sample into theappropriate injection port in accordance with the retention time window(block 1066).

After the aliquot of the prepared sample is injected into theappropriate injection port, the aliquot will traverse the multi-portvalve, loop, and columns as was described in greater detail above. Theanalytes eluted from the columns are then provided to the massspectrometer for analyte quantification. Once the analyte(s) of interestis/are detected and measured by the ion detector of the massspectrometer, the sample preparation controller will determine how toprocess and report the measured data.

FIG. 31 is a flowchart 1070 illustrating a sequence of operations forthe processing and reporting various sample types consistent withembodiments of the present invention. The sample preparation controllermay determine whether the measured result data has been received (block1072). If the result data has been input into the sample preparationcontroller (“Yes” branch of decision block 1072), then the samplepreparation controller will calculate a response (block 1076); if noresult data is received (“No” branch of decision block 1072), then thesequence of events returns to block 1072.

After the response is calculated (block 1076), the sample preparationcontroller determines the type of sample that is being analyzed (block1078). If the sample was a known standard for generating a calibrationcurve (“Calibration” branch of decision block 1078), then the samplepreparation controller will determine whether all result data for allcalibration tests has been received, i.e., whether allconcentrations/dilutions of the known calibration standard have beenanalyzed by the mass spectrometer (block 1080). When all calibrationtests have been analyzed and the results received (“Yes” branch ofdecision block 1080), the sample preparation controller will calculatethe calibration curve (block 1082). Calculation of the calibration curveis in accordance with a mathematical model, which for example, mayinclude any degree of polynomial, exponential, logarithmic, natural log,or other type of curve, where the calibration curve is fit to the resultdata. When there is less than a required number of result data pointsfor one or more calibration standards (“No” branch of decision block1080), the sample preparation controller returns to block 1072.

Once the calibration curve has been calculated, the sample preparationcontroller will make a determination as to whether the calibration curveis acceptable (block 1084). The criteria for determining whether acalibration curve is acceptable may be based on any known statisticalmodel, where the degree of fit between the calibration curve, ascompared with the result data, is quantified. One exemplary manner ofanalysis includes a statistical regression where the calibration curvemust “fit” the result data within a designated standard deviation. Ifthe fit of the calibration curve to the result data is acceptable (“Yes”branch of decision block 1084), then the calibration curve isautomatically accepted (block 1086). If the calibration curve is notacceptable (“No” branch of decision block 1084), then the samplepreparation controller will output “Calibration Curve Acceptable?”(block 1088) for the user to make a manual determination on thecalibration curve.

Returning again to the determination of the sample type (block 1078), ifthe determination is that the sample is a specimen (“Specimen” branch ofdecision block 1078), then the sample preparation controller willcalculate the sample results using the current calibration curve (block1090). The current calibration curve may be any calibration curve thathas been previously automatically or manually accepted and subsequentlyused without error or invalidation. In some embodiments, the calculationof sample results includes the evaluation of the sample results againstthe calibration curve and the extrapolation of the analyteconcentration, as is known in the art.

The sample preparation controller then makes a determination of whetherthe calculated sample result is acceptable in view of the currentcalibration curve (block 1092). In practice, this may include anevaluation as to whether the sample results fall within the bounds ofthe calibration curve, i.e., the minimum and maximum extremeconcentrations of the calibration standards analyzed in calculating thecurrent calibration curve. If the analyte response for the sample fallsjust beyond the bounds of the current calibration curve (for example,within a predetermined percentage or duration), then a new sample fromthe specimen may be acquired and prepared in accordance with the assaybut at a new dilution factor and the present analyte response discarded.If the analyte response falls outside of the predetermined percentage ordeviation, then a new calibration test may be triggered and the presentanalyte response discarded. If no analyte response results or a responsefrom an internal standard falls outside a predetermined bound, then anerror may be indicated and the present analyte response discarded. Ifthe calculated sample result is acceptable (“Yes” branch of decisionblock 1092), then the sample preparation controller will automaticallyreserve the sample results for the next control review (block 1094). Inthis way, the performance and/or operation of the system may beassessed. If the system were to fail a subsequent control analysis, thenthe accuracy and/or the precision of the sample results calculated anddeemed acceptable (blocks 1090 and 1092, respectively) may be calledinto question. If the calculated sample result is not acceptable (“No”branch of decision block 1092), then the sample preparation controllerwill output “Sample Result Acceptable?” (block 1096) for the user tomake a manual determination.

Once again returning to the determination of the sample type (block1078), if the determination returns that the sample is a known standardsample for performing a control test (“QC” branch of decision block1078), then the sample preparation controller will calculate the controlresults using the current calibration curve (block 1096). Thiscalculation may be performed in a manner that is similar to the methoddescribed above with reference to the specimen (see block 1090). Thesample preparation controller will then determine whether all controlresults for all control tests has been received, i.e., whether allconcentrations/dilutions of the known control standards have beenanalyzed by the mass spectrometer (block 1098). When all control testshave been received (“Yes” branch of decision block 1098), the samplepreparation controller will analyze all control results (block 1100). Ifnot all control tests are received (“No” branch of decision block 1098),then the sequence returns to block 1072.

After analysis of the control results (block 1100), the samplepreparation controller will determine whether the control results areacceptable (block 1102). The criteria used for this determination mayvary and one of ordinary skill in the art will readily appreciate theparticular statistical analysis that will best make the determination.According to one embodiment, the sample preparation controller may makethe determination on the Westgard Rules (J O Westgard, et al. “Amulti-rule Shewhart chart for quality control in clinical chemistry.”Clin. Chem. 1981; 27:493-550.), which uses a combination of criteria,referred to a control rules, for determining whether an analyticalsystem is “in-control” or “out-of-control.” The rules may include: (1)review result data when a single control measurement exceeds the mean±3σ(where 3σ is the third standard deviation); (2) reject all data when asingle control measurement exceeds the mean±2σ; (3) reject all data whena run of two consecutive control measurements exceed the mean±2σ; (4)reject all data when a first control measurement within a group exceedsthe mean±2σ and a second control measurement within the group exceedsthe mean±2σ in the opposite direction as the first control measurement;and (5) reject all data when first, second, third, and fourth controlmeasurements (in succession) are on the same side of the mean±1σ. Otherrules are known and may be included as appropriate.

If the control results are acceptable (“Yes” branch of decision block1102), then the sample preparation controller will automatically acceptthe control results (block 1104) and release the sample results forreporting (block 1106). The sample report is then sent by the samplepreparation controller (1108). Further, if further analysis of thesample is not required, the vessel may be discarded. If the controlresults are not acceptable (“No” branch of decision block 1102), thenthe sample preparation controller will output “control ResultsAcceptable?” (block 1110) for the user to make a manual determination.

In certain assay embodiments, for example for testing microbiologicalsamples, two or more sample analysis methods may be performed on asingle specimen. For example, a first sample analysis method may beperformed to obtain a full identification of the analyte or set ofanalytes in the sample, acquiring data over the entire sample elutiontime window. Following analysis of the full identification data, thesample analysis controller may return a result indicating theidentification of the analyte or microorganism (based upon a set ofanalytes) in the sample. The resulting identification may result in arequest for a second sample analysis method, for example, to performantibiotic susceptibility testing (“AST”) on the identified sample, forexample, by incubating one or more aliquots of the sample in thepresence of one or more antibiotics. In the case of AST testing, thesecond sample analysis method may acquire data only over a specifiedretention time window specific to the identified sample.

The sample preparation controller, in addition to normal functions tooperate the sample preparation station and coordinate with the sampleanalysis controller to perform a test, is configured to performhousekeeping routines to consolidate vessel racks and clear the samplepreparation station of samples that are no longer necessary. Inparticular, FIG. 32 is a flowchart 1120 for the sample preparationcontroller to consolidate vessels in vessel racks, if used, andconsistent with embodiments of the present invention utilizing vesselracks. In particular, the sample preparation station determines whetherany vessel rack in the sample preparation station has less than threevessels (block 1122). When there is no vessel rack in the samplepreparation station with less than three vessels (“No” branch ofdecision block 1122), the sequence of operations may end. Otherwise,when there is a vessel rack in the sample preparation station with lessthan three vessels (“Yes” branch of decision block 1122), the samplepreparation station selects that vessel rack with less than threevessels (block 1124) and determines whether there is vacant space (e.g.,a place for an additional vessel) in any other vessel racks in thesample preparation station (block 1126). When there is vacant space inone or more vessel racks (“Yes” branch of decision block 1126), one ormore vessels are moved from the selected vessel rack to the one or morevessel racks with vacant space (block 1128). When there is no vacantspace in one or more vessel racks (“No” branch of decision block 1126),or in response to moving vessels from a selected vessel rack to one ormore vessel racks with vacant space (block 1128), the sequence ofoperations may end.

FIG. 33 is a flowchart 1130 for the sample preparation controller todetermine whether specimens may be discarded from the sample preparationstation consistent with embodiments of the present invention. The samplepreparation station may initially select a specimen to analyze forhousekeeping (block 1132) and determine whether all test for thatspecimen have been completed (block 1134). When all tests for thespecimen have not completed (“No” branch of decision block 1134), thesample preparation controller selects the next specimen to analyze forhousekeeping (block 1136) and the sequence of operations proceeds backto block 1132. However, when all tests for the specimen have beencompleted (“Yes” branch of decision block 1134), the sample preparationcontroller indicates, or stores an indication, that the specimen may bediscarded (block 1138) and the sequence of operations proceeds back toblock 1136. In some embodiments, a user may remove specimens that may bediscarded, while in alternative embodiments the sample preparationstation may be configured to move such specimens to the wastereceptacle.

FIGS. 34A-34B are a flowchart 1310 for a method of booting up the systemconsistent with embodiments of the present invention. The systemreceives a command from the user to turn on (block 1320), which causesthe client and the service to initiate a booting sequence (block 1322).Once the sample preparation controller is sufficiently booted, thesample preparation controller transmits a command (block 1326) to startthe pumps (block 1324), which may include starting a turbo pump (i.e., apump that is internal to the mass spectrometer) and a roughing pump(i.e., a pump that is external to the mass spectrometer). A statusupdate may be output indicating that the system is booting (block 1328).If a secured system is used, the sample prep controller may output alogon command from an authorized user (block 1330).

During the method of booting up, the sample preparation controllermonitors the vacuum pressures and functionality of the pumps over aperiod of time and reports a status of the same (block 1334). While thetime period may vary, generally a period ranging from about 4 hours toabout 8 hours is appropriate. If the pump stability time is not complete(“No” branch of decision block 1332), then the sample prep controllercontinues to monitor the received pump status and operation parameters(block 1332). Otherwise (“Yes” branch of decision block 1332), themethod continues and an output may be displayed indicating that thepumps are ready (block 1336).

The sample prep controller may then proceed to monitor the status of oneor more fluid levels within the system. In that regard, the sample prepcontroller may query as to whether the mobile phase levels are ok (block1338). If the mobile phase levels are low or not available (“No” branchof decision block 1338), then a mobile phase level error output (block1340) is provided. The error may include specific instructions as towhich mobile phase levels are low, other errors, as appropriate, and/orsolutions to the error. If the mobile phase levels are ok (“Yes” branchof decision block 1338), then the method continues by checking the wastecontainer level (block 1342). If the waste container is absent or full(i.e., beyond a threshold volume) (“No” branch of decision block 1342),then a waste container level error output (block 1344) is provided.Again, the error may provide a suitable solution to the user. If thewaste container levels are ok (“Yes” branch of decision block 1342),then the method may continue by checking the needle wash buffer level(block 1346). If the needle wash buffer levels are low or not available(“No” branch of decision block 1346), then a needle wash level erroroutput (block 1348) is provided and, optionally, include one or moreinstructions and/or solutions. If the needle wash levels are ok (“Yes”branch of decision block 1346), then the sequence of operationscontinues.

The sample preparation controller may then transmit a command (block1352) to check the column life remaining for a particular loaded columncartridge (block 1350). Column life may be determined by one or morefactors, including, for example, a number of samples run, a total timeof use, a total volume injected, and so forth. Based on one or more ofthese factors, a column life remaining is provided (block 1354) and, ifthe column life is below a predetermined threshold (“Yes” branch ofdecision block 1356), then the sequence may end. Otherwise, a columnlife error output (block 1358) may be output and the method may returnto block 1350 and the output may include information such as whichcartridge to replace.

FIGS. 35A-35B are a flowchart 1360 for a method of starting the systemand entering an idle state in preparation for receiving a specimenconsistent with embodiments of the present invention. In that regard, astartup command is received (block 1362). The sample prep controllerthen transmits a command (block 1366) to prime the pumps (block 1364)and monitors the status thereof. If the pumps are not primed (“No”branch of decision block 1368) as indicated by a received pump status(block 1370), then a pump error (block 1372) is provided and the systemcontinues to monitor the status. Otherwise (“Yes” branch of decisionblock 1368), the method continues and the sample preparation controllermay transmit a command (block 1376) to prime the syringes and the probewash (block 1374). If the syringe and/or the probe wash are not primed(“No” branch of decision block 1378) as indicated by a received syringeand probe wash status (block 1380), then a syringe and probe wash error(block 1382) is provided and the system continues to monitor the status.If the syringe and probe wash are primed (“Yes” branch of decision block1378), then the method may continue.

The sample prep controller may then transmit a command (block 1386) toperform a method for checking the calibration of the mass spectrometer(block 1384). In that regard, a mass spectrometer calibration solutionis injected and analyzed in accordance with a selected assay. Thecalibration solution may include a number of known analytes, at knownconcentrations, and that are suitable for comparison with the currentcalibration curve. The results of the analysis are then compared againstthe known values for determining whether the current calibration remainsvalid. If the analysis indicates that the calibration has passed (“Pass”branch of decision block 1388) as determined from a returned status ofthe calibration check (block 1390), then the method continues and thesystem may enter an idle state, which is described in greater detailbelow. If the analysis indicates that the calibration has failed (“Fail”branch of decision block 1388), then the sample preparation controllermay transmit a command (block 1394) for performing a mass spectrometercalibration method (block 1392). The calibration method includesinjecting a number of solutions, each containing a varying amount of oneor more analytes, for generating a calibration curve in manner that isdiscussed in greater detail below. If the mass spectrometer calibrationpasses (“Pass” branch of decision block 1396) as indicated by the statusreceived (block 1398), then the system may enter an idle state; however,if the calibration fails (“Fail” branch of decision block 1396), then amass spectrometer calibration error is output (block 1400).

To enter the idle state, the sample preparation controller transmits acommand (block 1404) to run a double blank method for all channels thatare presently in use (block 1402). In that regard, a blank injection(also referred to as a “fake” injection) is made and the analyzed andthen repeated. Once the second blank injection has been analyzed and thedouble blank method is complete (“Complete” branch of decision block1406) as determined from a received double blank status (block 1408),then the system returns to block 1406 may alert the user of the idlestate status and output an idle indication (block 1410). If the doubleblank method is not complete (“Pending” branch of decision block 1406),then the system waits until the determination is “Yes.”

With the system in the idle state, the sample prep controller maycontinue to transmit commands (block 1404) for double blank methods(block 1402) until a start, standby, or shutdown command is received. Inthat regard, the standby method is described with reference to FIG. 36(“Standby” branch of decision block 1412), the shutdown method isdescribed with reference to FIG. 37 (“Shutdown” branch of decision block1412), and the start method may include any one of the methods asprovided in FIGS. 17-33 (“Start” branch of decision block 1412).

FIG. 36 is a flowchart 1420 illustrating a method of entering a standbystate consistent with embodiments of the present invention. The systemmay receive a command to enter a standby state (block 1422), whichcauses the sample preparation controller to transmit a command (block1426) to initiate a standby method (block 1424). The system may thenindicate that it is entering the standby mode (block 1428) and thenprepares and analyzes a single sample according to a standby assayprocedure (block 1430). If the standby method is determined to beincomplete (“No” branch of decision block 1432) from the receivedstandby method status (block 1434), then the system continues to waitand monitor the standby method. If the standby method is completed(“Yes” branch of decision block 1432), then a standby output is provided(block 1436) and remains until a wake up command is received (block1438). So long as no wake up command is received (“No” branch ofdecision block 1438), then the system remains in standby. If a wake upcommand is received (“Yes” branch of decision block 1438), such asloading a specimen or the user indicating the system should start orshutdown, then the method may initiate the start up method 1440, as wasdescribed in the flowchart 1360 of FIG. 35. In standby, the pumps maynot be operating, the column heaters may be off; the gas pressure may beoff, the capillary temperature of the mass spectrometer may be decreasedto about 200° C., and/or the reagent cooling may remain on.

FIG. 37 is a flowchart 1450 illustrating a method of shutting down thesystem consistent with embodiments of the present invention. The systemreceives a shutdown command (block 1452), transmits a command (block1456) to initiate a shutdown procedure (block 1454), and may output ashutting down indication (block 1458). This may include terminating theoperation of the pumps and stopping all fluid flows while maintainingheating and cooling in those portions of the system as appropriate. Whenthe shutdown procedure is complete (“Yes” branch of block 1460) asdetermined by a received shutdown status (block 1462), then the systemmay indicate that shutdown is complete (block 1464). Otherwise, thesystem continues to monitor the shutdown status (“No” branch of decisionblock 1460).

FIG. 38 illustrates a fluid system for managing one or more fluid levelswithin the system 10 (FIG. 1A) and in accordance with one embodiment ofthe present invention. In that regard, two load cells 1470, 1472 may beoperably coupled to the sample preparation controller 22 or anothercontroller as appropriate. Each load cell 1470, 1472 is associated witha fluid container 1474, 1476 containing the same fluid 1478 therein. Asshown, a first fluid container 1474 may be larger volume as compared tothe second fluid container 1476 such that the first fluid container 1474is a fluid supply and the second fluid container 1476 is an auxiliaryfluid supply. While not required, the second fluid container 1476 may beconfigured to contain a smaller volume of the fluid 1478 as compared tothe first container 1474 so as to be stored with the system 10 (FIG. 1A)under the cover 16 (FIG. 1A) and yet coupled to an external andaccessible source. A first fluid line 1480 extends between the firstfluid container 1474 and the second fluid container 1476, and a secondfluid line 1482 extends between the second fluid container 1476 and isfluidically coupled to the system 10 (FIG. 1A) via a solenoid valve1484. The fluid 1478 of the second fluid container 1476 is under partialvacuum as the second fluid container 1476 is coupled to a vacuum chamber1486 and a vacuum pump 1488 via a separate vacuum line 1490.

In use, as the fluid 1478 is removed from the second fluid container1476 during the preparation and/or analysis of one or more samples inaccordance with one or more assays, the weight of the second fluidcontainer 1476 with the fluid 1478 decreases, as determined by thesecond load cell 1472 at the sample preparation controller 22. When theweight of the second fluid container 1476 with the fluid 1478 fallsbelow a first threshold value (e.g., an indirect measure of a minimumfluid volume), then the vacuum pump 1488 is activated to draw a vacuumon the vacuum chamber 1486. Because the vacuum chamber 1486 is coupledto the sealed second fluid container 1476, the vacuum pump 1488 alsodraws a vacuum on the second fluid container 1476. With sufficientvacuum pressure generated within the second fluid container 1476, thefluid 1478 may be withdrawn from the first fluid container 1474, throughthe first fluid line 1480, and into the second fluid container 1476. Thefluid 1478 will continue to transfer the fluid 1478 from the first fluidcontainer 1474 to the second fluid container 1476 until the weight ofthe second fluid container 1476 with the fluid 1478 therein reaches asecond threshold value (e.g., an indirect measure of a maximum fluidvolume). At that time, the sample preparation controller 22 sends acommand to the vacuum pump 1488 to terminate operation, the vacuumwithin the vacuum chamber 1486 decreases, and the fluid transfer stops.

Although not shown, one having ordinary skill in the art will appreciatethat the sample preparation controller may further control the samplepreparation station to discard vessels of prepared samples that havebeen analyzed. As such, the sample preparation controller may firstdetermine whether the prepared samples in such vessels have beenanalyzed, and whether an analysis of that prepared sample should bere-performed. When re-performance of an analysis is unnecessary, thevessel with the prepared sample may be discarded to a waste receptacle.Otherwise, the vessel may be kept in the sample preparation station.

EXAMPLE 1

Turning now to FIG. 39A, five chromatograms 1200, 1202, 1204, 1206, 1208corresponding to each of five analytes contained within a second sampleare schematically shown as would be detected on a LC-MS system. Achromatogram is a graphical representation of the time dependent totalion count (“TIC”) for each analyte of interest measured at the iondetector 150 (FIG. 4B). Accordingly, the chromatogram includes retentiontime along the x-axis, TIC along the y-axis, and m/z along the z-axis.In the particular example, the first, second, and third chromatograms1200, 1202, 1204 correspond to first, second, and third analytes thatelute substantially simultaneously off of the columns 114, 116 (FIG. 7A)during a first elution window. The fourth and fifth chromatograms 1206,1208 correspond to fourth and fifth analytes, respectively, which eluteat later times (second and third elution windows, respectively).

The LC-MS system is capable of scanning at a rate that ranges from 2000amu/sec to about 5000 amu/sec, or said another way, at about 10 SRM/secto about 50 SRM/sec, where SRM represents an observed “selected reactionmonitoring,” transition. Accordingly, and for purposes of example only,a two second elution window during which the chromatographic peakcontaining the analytes of interest is delivered by the LC to the massspectrometer 120 (FIG. 4B) could be scanned 20 to 50 times during theelution window. This replication of measurements improves thesignal-to-noise ratio and may result in greater sensitivity for lowconcentration analytes. However, this replication of measurements alsorequires large data storage capabilities. In order to reduce the amountof information saved and transmitted from the sample analysis controller26 (FIG. 2) to the sample preparation controller 22 (FIG. 2) thereplicated TIC data measured by the ion detector 150 (FIG. 4B) may besampled, filtered, or otherwise compressed into raw data fortransmission from the sample analysis controller 26 (FIG. 2) to thesample preparation controller 22 (FIG. 2). For example, sampling of thechromatogram for each analyte may include saving an m/z value every 30milliseconds. In addition to this sampled data, the raw data for eachchromatogram may include the m/z value, an integral of the chromatogram,and the maximum TIC. This raw data, along with a sample/test identifierand the elution window are sent to the sample preparation controller 22(FIG. 2) for further post processing.

The raw data, as shown in FIG. 39B, may be in a tabular format or anyother human perceivable format or, alternatively and/or additionally theraw data may be formatted for electrical transmission to the samplepreparation controller 22 (FIG. 2) for additional post processing.

The raw data may then be evaluated against an appropriate calibrationcurve for the corresponding analyte. The calibration curve may beconstructed in a known manner. Briefly, this includes the preparationand analysis of two or more samples containing a known but varyingamount of the analyte. The response of the ion detector 150 (FIG. 4B) isthen recorded for the known samples and plotted against concentration.For simplicity, detection and reporting on the internal standard is notshown. A mathematical model is fit to the known sample data. Exemplarycalibration curves are shown in FIGS. 39C-39E as follows: FIG. 39Cillustrates a linear response with concentration of analyte 1; FIG. 39Dillustrates a negative exponential response with concentration ofanalyte 2; and FIG. 39E illustrates a positive exponential response withconcentration of analyte 3. One of ordinary skill in the art wouldreadily appreciate that a wide range of mathematical models may be usedto correlate the analyte response with concentration and the manner bywhich the model may be determined, for example, a least squares fitregression.

With the mathematical model of the calibration curve determined, the rawdata for the prepared sample, i.e., an unknown sample, is evaluated inview of the calibration curve. Accordingly, the raw data representingthe response of the appropriate analyte of the prepared sample isplotted against the calibration curve. The resultant concentration ofthe analyte within the prepared sample may be extrapolated from thecalibration curve. Finally, and from the dilution factors associatedwith the test, the concentration of the analyte in the original specimenmay be determined from the prepared sample in accordance with:

C1V1=C2V2

wherein, C1 is the concentration of the analyte within the preparedsample, V1 is the volume of the prepared sample, V2 is the volume of thespecimen, and C2 is the determinable concentration of the analyte in thespecimen.

It will further be appreciated that if the response of the analyte ofthe prepare sample falls outside of the range of concentrations testedfor a particular calibration curve, then the calibration curve may beinvalid and inappropriate for evaluation of the prepared sample.

EXAMPLE 2

FIG. 40 illustrates TIC for all m/z values measured, in toto, at the iondetector 150 (FIG. 4B) of the mass spectrometer 120 (FIG. 4B) for aplurality of prepared samples, ad infinitum, where the prepared samplesundergo a multiplexing protocol and are scheduled as described in detailpreviously. The peaks represent one or more gas phase ions correspondingto one or more analytes eluting off of the columns 114, 116 (FIG. 7A).In this way, and as clearly demonstrated in this figure, the resourcesof the mass spectrometer 120 (FIG. 4B) are maximized as compared toconventional methodologies where one sample must completely elute from acolumn 114, 116 (FIG. 7A) and be analyzed before a second sample may beinjected into the column 114, 116 (FIG. 7A) for analysis.

As also shown in FIG. 40, each time a new sample is injected into thecolumns 114, 116 (FIG. 7A), a signal may be generated to indicate azeroing of the retention time window and the start of a new analysis.The signal may be generated when the valve 126 a, 126 b (FIG. 4B) isswitched from the “fill in loop” position to the “in-line” position.

Further, and as is clearly demonstrated in the figure, the analytescontained within any one particular sample may not necessarily be thesame in type or in quantity as analytes contained in any one of theother samples.

While the present invention has been illustrated by description ofvarious embodiments and while those embodiments have been described inconsiderable detail, it is not the intention of applicant to restrict orin any way limit the scope of the appended claims to such details.Additional advantages and modifications will readily appear to thoseskilled in the art. The invention in its broader aspects is thereforenot limited to the specific details and illustrative examples shown anddescribed. Accordingly, departures may be made from such details withoutdeparting from the spirit or scope of applicants' invention.

What is claimed is:
 1. An automated sample preparation and analysissystem, comprising: a sample preparation system configured to prepare asample of a specimen in accordance with an assay selected from adatabase containing a plurality of unique assays; a sample analysissystem configured to analyze the prepared sample in accordance with theselected assay; and a controller configured to sequence the sample forpreparation by the sample preparation system and further configured tosequence the prepared sample for analysis by the sample analysis system.2. The automated sample preparation and analysis system of claim 1,wherein the controller is configured to control the operation of thesample preparation system.
 3. The automated sample preparation andanalysis system of claim 1, wherein the sequence for the preparation ofthe sample is dependent on at least one of the order of arrival ofrespective specimens at the sample preparation and analysis system, orthe priority of respective specimens.
 4. The automated samplepreparation and analysis system of claim 3, wherein the sequence for thepreparation of the sample is further dependent on at least one of atarget time to result for a respective sample, a remaining target timeto result for a respective sample, the number of samples in the samplepreparation and analysis system, the number of samples associated withthe each of the unique assays, or reconfiguration of the sample analysissystem required for selected assays preceding and following a respectiveselected assay.
 5. A method of preparing and analyzing samples takenfrom specimens, comprising: sequencing the samples for preparation inaccordance with respective assays selected from a database containing aplurality of unique assays; and sequencing the analysis of the preparedsamples in accordance with the respective selected assay.
 6. The methodof claim 5 further comprising: preparing a first sample in accordancewith a first selected assay; and re-sequencing at least one of thesamples for preparation.
 7. The method of claim 5 further comprising:reserving at least one component of a sample preparation system forpreparing a first sample in accordance with a respective selected assay.8. The method of claim 5, wherein sequencing the samples for preparationincludes: determining a target time to result for each of the samples;and ordering the samples for preparation by their respective targettimes to result.
 9. The method of claim 5, wherein sequencing thesamples for preparation includes: determining a target time to preparefor each of the samples; and ordering the samples for preparation bytheir respective target times to prepare.
 10. The method of claim 5,wherein sequencing the prepared samples for analysis includes:determining a target time to result for each of the prepared samples;and ordering the prepared samples for analysis by their respectivetarget times to result.
 11. The method of claim 5 further comprising:overriding at least a portion of the sequencing of the samples toprepare a known control sample.
 12. The method of claim 5 furthercomprising: overriding at least a portion of the sequencing of theprepared samples to analyze a prepared known control sample.
 13. Themethod of claim 5, further comprising: overriding at least a portion ofthe sequencing of the samples to prepare a calibration sample.
 14. Themethod of claim 5, further comprising: overriding at least a portion ofthe sequencing of the prepared samples to perform a calibration of ananalyzer used to analyze the prepared samples.
 15. The method of claim5, further comprising: overriding at least a portion of the sequencingof the samples to prepare a quality control sample.
 16. The method ofclaim 5, further comprising: overriding at least a portion of thesequencing of the prepared samples to perform a quality control of ananalyzer used to analyze the prepared samples.
 17. The method of claim5, wherein the sequencing of the samples is dependent on at least one ofthe order of arrival of respective samples at the sample preparation andanalysis system, or the priority of respective samples.
 18. The methodof claim 17, wherein the sequencing of the samples is further dependenton at least one of a target time to result for a respective sample, aremaining target time to result for a respective sample, the number ofsamples in the sample preparation and analysis system, the number ofsamples associated with the each of the unique assays, orreconfiguration of the sample analysis system required for selectedassays preceding and following a respective selected assay.
 19. Themethod of claim 5 further comprising: transporting a first preparedsample from a sample preparation system to a sample analysis system foranalysis in accordance with the respective predetermined assay; andre-sequencing at least one of the prepared samples remaining in thesample preparation system for analysis.
 20. The method of claim 5further comprising: reserving one or more components of a sampleanalysis system for analyzing a first prepared sample in accordance withrespective selected assay and the sequencing of the prepared samples.21. An automated sample preparation and analysis system, comprising: asample preparation system configured to prepare a sample of a specimenin accordance with an assay selected from a database containing aplurality of unique assays; a sample analysis system configured toanalyze the prepared sample in accordance with the selected assay; and acontroller configured to dynamically sequence the prepared sample foranalysis by the sample analysis system.
 22. The automated samplepreparation and analysis system of claim 21, wherein the controller isfurther configured to sequence the sample for preparation by the samplepreparation system.
 23. A method of preparing and analyzing samplestaken from specimens that are prepared for analysis, comprising:dynamically sequencing the analysis of the prepared samples inaccordance with respective assay selected from a database containing aplurality of unique assays.
 24. The method of claim 23 furthercomprising: sequencing samples for preparation in accordance with therespective selected assays.
 25. An automated sample preparation andanalysis system, comprising: a sample analysis system of the type thatincludes a mass spectrometer, the sample analysis configured to analyzea plurality of samples according to respective assays selected from adatabase containing a plurality of unique assays; and a samplepreparation system including a controller configured to sequence thesamples for analysis by the sample analysis system; wherein the sequenceof sample analysis is dependent on the order of arrival of each specimenat the automated sample preparation and analysis system, the prioritystatus of each sample, and at least one of a target time to result for asample, a target time to result for a selected assay for a sample, aremaining target time to result for a selected assay for a sample, thenumber of samples on-board the system, the number of samples utilizingthe same selected assay, or mass spectrometer reconfiguration requiredfor selected assays preceding and following a selected assay for aparticular sample.